Methods for modifying the production of a polypeptide

ABSTRACT

The present invention relates to methods for modifying the production of a polypeptide, comprising: (a) introducing a nucleic acid construct into a cell, wherein the cell comprises a DNA sequence encoding a polypeptide, under conditions in which the nucleic acid construct integrates into the genome of the cell at a locus not within the DNA sequence encoding the polypeptide to produce a mutant cell, wherein the integration of the nucleic acid construct modifies the production of the polypeptide by the mutant cell relative to the cell when the mutant cell and the cell are cultured under the same conditions; and (b) identifying the mutant cell with the modified production of the polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of pending U.S.application Ser. No. 08/713,312 filed on Sep. 13, 1996, whichapplication is fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods for modifying the production ofa polypeptide by a cell.

[0004] 2. Description of the Related Art

[0005] Several methods have been used to modify the production ofpolypeptides by mutagenizing cells. For example, the production ofproteins has been altered by producing mutant cells by classicalmutagenesis which involves treating cells with chemical, physical, andbiological agents as mutagenic (mutation inducing) agents to increasethe frequency of mutational events.

[0006] Production of proteins also has been modified by mutagenesis of acell with short sections of double-stranded DNA, consisting of more than2000 base pairs, called transposons which usually code for resistance toone or sometimes several antibiotics. Transposons are able to move orjump within the genome, even between a bacterial chromosome and aplasmid, and they are able to become integrated in a number of differentsites on the genome. An insertion of a transposon within a structuralgene interrupts the normal nucleotide sequence of the gene so that itcan no longer deliver the information for the synthesis of the normal,functional polypeptide (Seifert et al., 1986, Proceedings of theNational Academy of Sciences USA 83: 735-739). An insertion also maydisrupt a gene whose gene product is required for expression(Márquez-Magaña and Chamberlin, 1994, Journal of Bacteriology 176:2427-2434). In addition, Errede et al. (1980, Cell 22: 427-436) disclosethe insertion of a transposable element adjacent to the structural genecoding for iso-2-cytochrome c causing overproduction. Furthermore, WO96/29414 discloses that transposable elements may be constructedcontaining a transposon and a DNA sequence capable of regulating atargeted gene where upon introduction into a cell the transposableelement integrates into the genome of the cell in a manner whichregulates the expression of the gene.

[0007] A widely used method for increasing production of a polypeptideis amplification to produce multiple copies of the gene encoding thepolypeptide. For example, U.S. Pat. No. 5,578,461 discloses theinclusion via homologous recombination of an amplifiable selectablemarker gene in tandem with the gene where cells containing amplifiedcopies of the selectable marker can be selected for by culturing thecells in the presence of the appropriate selectable agent.

[0008] In addition, the production of polypeptides has been increased byreplacing one promoter with a different promoter or one signal peptidecoding region with another. See, e.g., U.S. Pat. No. 5,641,670.

[0009] Methods for altering gene expression by disrupting genes encodingvarious regulatory elements have also been described. For example, Tomaet al. (1986, Journal of Bacteriology 167: 740-743) showed that adeletion from −156 to −90 in the npr promoter region causedoverexpression of the neutral protease encoded by the npr gene. Pero andSloma (1993, In A. L. Sonensheim, J. A. Hoch, and R. Losick, editors,Bacillus subtilis and Other Gram-Positive Bacteria, pp. 939-952,American Society for Microbiology, Washington, D.C.) disclose thatmutating the sporulation gene spoOA results in deficient synthesis ofproteases and that mutations in the abrB gene restore synthesis.

[0010] The production of polypeptides also has been increased bydisrupting DNA sequences encoding a protease capable of hydrolyzing thepolypeptide under the conditions for producing the polypeptide.

[0011] The secretion of polypeptides has also been modified byoverproduction of secretion proteins (Ruohonen et al., 1997, Yeast, 13:337-351), and producing a super-secreting cell (U.S. Pat. No.5,312,735).

[0012] Methods for increasing the production of metabolites have alsobeen described. For example, WO 96/41886 discloses that increasedproduction of clavam produced by an organism having at least part of theclavam pathway and at least part of a cephalosporin pathway byinterfering with the conversion of L-lysine to L-alpha-aminoadipic acidin the cephalosporin pathway. WO 94/13813 discloses the disruption ofgene which encodes a protein which degrades betaine, an enzyme inducer.

[0013] It is an object of the present invention to provide new andimproved methodologies for altering production of polypeptides andmetabolites.

BRIEF SUMMARY OF THE INVENTION

[0014] The present invention is drawn to methods for modifying theproduction of a polypeptide by a cell. In the methods of the presentinvention, a nucleic acid construct is introduced into a cell whichcontains a DNA sequence encoding a specific polypeptide. The introducednucleic acid construct integrates into the host genome at a locus notwithin the DNA sequence of interest to produce a mutant cell. Theintegration of the nucleic acid construct into the locus modifies theproduction of the polypeptide by the mutant cell relative to the parentcell. Mutant cells are then identified in which the polypeptide'sproduction is modified by the mutant cell relative to the parent cell.Modification is determined by comparing production of the polypeptidewhen the mutant cell and the parent cell are cultured under the sameconditions.

[0015] An advantage of the present invention is that the mutation can berecovered and leads to a modification of the production of a polypeptideencoded by a DNA sequence which does not contain the mutation.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 is a restriction map of pJaL292.

[0017]FIG. 2 is a restriction map of pKS6.

[0018]FIG. 3 is a restriction map of pBANe13.

[0019]FIG. 4 is a restriction map of pBANe6.

[0020]FIG. 5 is a restriction map of pMHan37.

[0021]FIG. 6 is a restriction map of pBANe8.

[0022]FIG. 7 is a restriction map of pSO2.

[0023]FIG. 8 is a restriction map of pSO122 and shows the constructionof pDSY81 and pDSY82 from pSO122.

[0024]FIG. 9 is the nucleic acid sequence and the deduced amino acidsequence of the rescued locus of mutant Aspergillus oryzae DEBY599.3(SEQ ID NOS:9 and 10, respectively).

[0025]FIG. 10 is the nucleic acid sequence and the deduced amino acidsequence of the rescued locus of mutant Aspergillus oryzae DEBY10.3 (SEQID NOS: 16 and 17).

[0026]FIG. 11 is a restriction map of pJaL400.

[0027]FIG. 12 is the construction of pMT1935.

[0028]FIG. 13 is a restriction map of pJaL394.

[0029]FIG. 14 is a restriction map of pMT1931.

[0030]FIG. 15 is a restriction map of pMT1936.

[0031]FIG. 16 is the nucleic acid sequence and the deduced amino acidsequence of the rescued locus of mutant Aspergillus oryzae DEBY932 (SEQID NOS:25 and 26).

[0032]FIG. 17 is the nucleic acid sequence and deduced amino acidsequence of the rescued locus of mutant Aspergillus oryzae DEBY1058 (SEQID NOS:29 and 30).

[0033]FIG. 18 is a restriction map of pDSY161.

[0034]FIG. 19 is a restriction map of pDSY162.

[0035]FIG. 20 is the nucleic acid sequence of the rescued locus ofmutant Aspergillus oryzae 1204.3.3 (SEQ ID NO:34).

[0036]FIG. 21 is the nucleic acid sequence of the rescued locus ofmutant Aspergillus oryzae H603 (SEQ ID NO:39).

[0037]FIG. 22 is a restriction map of pGAG3.

[0038]FIG. 23 is a restriction map of pJaL389.

[0039]FIG. 24 is a restriction map of pJaL335.

[0040]FIG. 25 is a restriction map of pJaL399.

[0041]FIG. 26 is a restriction map of pDM176.

[0042]FIG. 27 is a restriction map of pHB218.

[0043]FIG. 28 is a restriction map of pSE39.

[0044]FIG. 29 is a restriction map of pDSY153.

[0045]FIG. 30 is a restriction map of pCaHj505.

[0046]FIG. 31 is a restriction map of pMStr107.

[0047]FIG. 32 is the nucleic acid sequence and deduced amino acidsequence of the rescued locus of mutant Aspergillus oryzae P4-8.1 (SEQID NOS:50 and 51).

[0048]FIG. 33 is the nucleic acid sequence and deduced amino acidsequence of the rescued locus of mutant Aspergillus oryzae P7-14.1 (SEQID NOS:56 and 57).

[0049]FIG. 34 is a restriction map of pMT1612.

[0050]FIG. 35 is the nucleic acid sequence and deduced amino acidsequence of the rescued locus of mutant Aspergillus oryzae DEBY7-17.2(SEQ ID NOS:63 and 64).

[0051]FIG. 36 is the nucleic acid sequence of the rescued locus ofmutant Aspergillus oryzae DEBY3-2.1 (SEQ ID NO:66).

[0052]FIG. 37 is the nucleic acid sequence of the rescued locus ofmutant Aspergillus oryzae DEBY5-7.1 (SEQ ID NO:71).

[0053]FIG. 38 is the nucleic acid sequence of the rescued locus ofmutant Aspergillus oryzae DEBY8-10.1 (SEQ ID NO:76).

DETAILED DESCRIPTION OF THE INVENTION

[0054] In a first embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0055] (a) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0056] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, not within a second DNAsequence encoding a protein that negatively regulates transcription,translation or secretion of the polypeptide, and not within a third DNAsequence encoding a protease capable of hydrolyzing the polypeptideunder the conditions; and

[0057] (ii) the mutant cell produces more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0058] (b) recovering the polypeptide.

[0059] A “protein that negatively regulates transcription” is definedherein as a repressor that negatively affects the process of RNAsynthesis by RNA polymerase to produce a single-stranded RNAcomplementary to a DNA sequence, or as a protein that degrades an enzymeinducer which is generally a chemical agent produced by a biosyntheticor catabolic pathway of a cell. The repressor consists of distinctdomains that are required for DNA-binding, transcription repression, andinducer or repressor binding.

[0060] A “protein that negatively regulates translation” is definedherein as a protein or a substance, the production of which is catalyzedby the protein, that negatively affects the process of protein synthesiscarried out by ribosomes which de-code the information contained in mRNAderived from transcription of a gene. For example, the substance may bea cap-dependent translation initiation factor, e.g., p20 (Altmann etal., 1997, EMBO Journal 16: 1114-1121); or a sex-lethal protein, e.g.,the sex-lethal protein of Drosophila which regulates the translation ofmsl-2 (Bashaw and Baker, 1997, Cell 89: 789-798).

[0061] A “protein that negatively regulates secretion” is defined hereinas a protein or a substance, the production of which is catalyzed by theprotein, that negatively affects the process of transferring a proteinmolecule through a membrane into (i) an intracellular compartment, e.g.,a vacuole or mitochrondrion, (ii) the periplasmic space, or (iii) theculture medium and, in eukaryotic cells, the process of vesiculartransport that ultimately results in exocytic release of secretedproteins from the cell. The secretory process oversees and promotescorrect protein folding, mediates any required post-translationalmodifications (such as glycosylation), and sorts, processes, and targetsproteins to specific cellular sites all at a rate consistent with thefunction of the cell as a whole. Such substances include a protein withCa²⁺-ATPase activity which upon inactivation increase levels of secretedheterologous or mutant proteins (for example, see Rudolph et al., 1989,Cell 58: 133-145); or the binding protein BiP which is an ATP-dependenthsp70-class chaperone found in the endoplasmic reticulum of eukaryoticcells which when decreased in mammalian cells through the use ofanti-sense RNA results in up to a three-fold increase in secreted levelsof a mutant protein (Dorner et al., 1988, Molecular and Cellular Biology8: 4063-4070). In a specific embodiment, the substance is a protein withATPase activity or the binding protein BiP.

[0062] In a second embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0063] (A) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0064] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, wherein the introduction ofthe nucleic acid construct disrupts a gene encoding an oxidoreductase, atransferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatoryor control sequences thereof, other than a gene encoding a proteasewhich is capable of hydrolyzing the polypeptide under the conditions;and

[0065] (ii) the mutant cell produces more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0066] (B) recovering the polypeptide.

[0067] A mutant cell that “produces” more of a polypeptide is definedherein as a cell from which more of the polypeptide is recoveredrelative to the parent cell.

[0068] In a third embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0069] (a) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0070] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, not within a second DNAsequence encoding a protein that negatively regulates transcription ofthe polypeptide, and not within a third DNA sequence encoding a proteasecapable of hydrolyzing the polypeptide under the conditions; and

[0071] (ii) the mutant cell expresses more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0072] (b) recovering the polypeptide.

[0073] In a fourth embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0074] (A) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0075] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, wherein the introduction ofthe nucleic acid construct disrupts a gene encoding an oxidoreductase, atransferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatoryor control sequences thereof, other than a gene encoding a proteasewhich is capable of hydrolyzing the polypeptide under the conditions;and

[0076] (ii) the mutant cell expresses more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0077] (B) recovering the polypeptide.

[0078] A mutant cell that “expresses” more of a polypeptide is definedherein as a cell that contains an increase in functional mRNA encodingthe polypeptide relative to the parent cell. It will be understood thatan increase in functional mRNA may result from an increase in theabsolute rate of transcription of the gene encoding the polypeptideand/or from alterations in post-transcriptional processing ormodification of the transcripts, including nuclear-cytoplasmic transportand/or cytoplasmic stabilization of the mRNA. Such mutant cells may beidentified using conventional techniques, including without limitationNorthern blot analysis, run-off transcription assays, and the like.

[0079] In a fifth embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0080] (a) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0081] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, not within a second DNAsequence encoding a protein that negatively regulates translation of thepolypeptide, and not within a third DNA sequence encoding a proteasecapable of hydrolyzing the polypeptide under the conditions; and

[0082] (ii) the mutant cell synthesizes more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0083] (b) recovering the polypeptide.

[0084] In a sixth embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0085] (A) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0086] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, wherein the-introduction ofthe nucleic acid construct disrupts a gene encoding an oxidoreductase, atransferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatoryor control sequences thereof, other than a gene encoding a proteasewhich is capable of hydrolyzing the polypeptide under the conditions;and

[0087] (ii) the mutant cell synthesizes more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0088] (B) recovering the polypeptide.

[0089] A mutant cell that “synthesizes” more of a polypeptide is definedherein as a cell that accumulates larger amounts of the polypeptiderelative to a parent cell. Accumulation refers to the total amount ofthe polypeptide in the culture as a whole, i.e., in both intracellularand extracellular compartments taken together. Such mutant cells may beidentified using any suitable technique, including without limitationpulse-labelling or steady-state labelling using radiolabelled aminoacids; immunoblot analysis of cell and medium fractions using anantibody specific to the polypeptide; assays of biological activity;separation by conventional chromatographic methods; and the like.

[0090] In a seventh embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0091] (a) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0092] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, not within a second DNAsequence encoding a protein that negatively regulates secretion of thepolypeptide, and not within a third DNA sequence encoding a proteasecapable of hydrolyzing the polypeptide under the conditions; and

[0093] (ii) the mutant cell secretes more of the polypeptide than theparent cell when both cells are cultivated under the conditions;

[0094] (b) recovering the polypeptide.

[0095] In an eighth embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0096] (A) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0097] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, wherein the introduction ofthe nucleic acid construct disrupts a gene encoding an oxidoreductase, atransferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatoryor control sequences thereof, other than a gene encoding a proteasewhich is capable of hydrolyzing the polypeptide under the conditions;and

[0098] (ii) the mutant cell secretes more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0099] (B) recovering the polypeptide.

[0100] A mutant cell that “secretes” more of a polypeptide is definedherein as a cell in which the amount of the polypeptide released intothe extracellular medium is increased relative to the parent cell. Suchmutant cells may be identified using, e.g., pulse-chase labelling inconjunction with immunoprecipitation to quantify the proportion of thenewly synthesized polypeptide that is externalized as well as theabsolute amount released in the mutant cell relative to the parent cell.Immunoblot analysis, biological activity assays, and physical-chemicalseparation methods may also be used to quantify the absolute amounts ofthe polypeptide released in mutant vs. parent cells.

[0101] In a ninth embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0102] (a) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0103] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the random integrationof a nucleic acid construct into the genome of the parent cell at alocus wherein the nucleic acid construct is not homologous with thelocus and wherein the locus is not within the first DNA sequence norwithin a second DNA sequence encoding a protease capable of hydrolyzingthe polypeptide under the conditions; and

[0104] (ii) the mutant cell produces more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0105] (b) recovering the polypeptide.

[0106] In a tenth embodiment, the present invention relates to methodsof producing a polypeptide, comprising

[0107] (a) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0108] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence and a second DNA sequenceencoding a protein that positively regulates transcription, translationor secretion of the polypeptide; and

[0109] (ii) the mutant cell produces less of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0110] (b) recovering the polypeptide.

[0111] A mutant cell that “produces” less of a polypeptide is definedherein as a cell from which less of the polypeptide is recoveredrelative to the parent cell.

[0112] A “protein that positively regulates transcription” is definedherein as an activator or an inducer that positively affects the processof RNA synthesis by RNA polymerase to produce a single-stranded RNAcomplementary to a DNA sequence. The activator consists of distinctdomains that are required for DNA-binding, transcription activation, andinducer or repressor binding. An inducer is generally a chemical agentproduced by a biosynthetic or catabolic pathway of a cell. In a specificembodiment, the substance is an activator or an inducer.

[0113] A “protein that positively regulates translation” is definedherein as a protein or a substance, the production of which is catalyzedby the protein, that positively affects the process of protein synthesiscarried out by ribosomes which de-code the information contained in mRNAderived from transcription of a gene. In a specific embodiment, thesubstance is an initiation factor or an elongation factor.

[0114] A “protein that positively affects secretion” is defined hereinas a protein or a substance, the production of which is catalyzed by theprotein, that positively affects the process of transferring a proteinmolecule through a membrane into (i) an intracellular compartment, e.g.,a vacuole or mitochrondrion, (ii) the periplasmic space, or (iii) theculture medium or positively affects vesicular transport as describedabove. Such substances include folding proteins, e.g., protein disulfideisomerase and peptidyl prolyl isomerase isoforms; chaperones, e.g., heatshock proteins, signal recognition particles, PrsA, SecD, SecF, and BiP;translocating chain-associating membrane proteins (TRAM); translocasecomplexes; and processing enzymes, e.g., glycosylating enzymes; signalpeptidases; pro region peptidases. In a specific embodiment, thesubstance is a folding protein, a chaperone, a signal recognitionparticle, PrsA, SecD, SecF, BiP, a translocating chain-associatingmembrane, a translocase complex, or a processing enzyme.

[0115] Other embodiments of the present invention relate to methods forproducing polypeptides as described in the tenth embodiment, except thatthe mutant cells express, synthesize or secrete less of the polypeptidethan the parent cell when both cells are cultivated under theconditions.

[0116] A mutant cell that “expresses” less of a polypeptide is definedherein as a cell that contains a decrease in functional mRNA encodingthe polypeptide relative to the parent cell. It will be understood thata decrease in functional mRNA may result from a decrease in the absoluterate of transcription of the gene encoding the polypeptide and/or fromalterations in post-transcriptional processing or modification of thetranscripts, including nuclear-cytoplasmic transport and/or cytomplasmicstabilization of the mRNA. Such mutant cells may be identified usingconventional techniques, including without limitation Northern blotanalysis, run-off transcription assays, and the like.

[0117] A mutant cell that “synthesizes” less of a polypeptide is definedherein as a cell that accumulates smaller amounts of the polypeptiderelative to a parent cell. Accumulation refers to the total amount ofthe polypeptide in the culture as a whole, i.e., in both intracellularand extracellular compartments taken together. Such mutant cells may beidentified using any suitable technique, including without limitationpulse-labelling or steady-state labelling using radiolabelled aminoacids; immunoblot analysis of cell and medium fractions using anantibody specific to the polypeptide; assays of biological activity;separation by conventional chromatographic methods; and the like.

[0118] A mutant cell that “secretes” less of a polypeptide is definedherein as a cell in which the amount of the polypeptide released intothe extracellular medium is decreased relative to the parent cell. Suchmutant cells may be identified using, e.g., pulse-chase labelling inconjunction with immunoprecipitation to quantify the proportion of thenewly synthesized polypeptide that is externalized as well as theabsolute amount released in the mutant cell relative to the parent cell.Immunoblot analysis, biological activity assays, and physical-chemicalseparation methods may also be used to quantify the absolute amounts ofthe polypeptide released in mutant vs. parent cells.

[0119] The present invention also relates to methods of producing ametabolite, comprising

[0120] (A) cultivating a mutant cell under conditions conducive forproduction of the metabolite, wherein

[0121] (i) the mutant cell is related to a parent cell, which comprisesone or more first DNA sequences encoding first polypeptides in thebiosynthetic pathway of the metabolite, by the introduction of a nucleicacid construct into the genome of the parent cell at a locus which isnot within (a) the first DNA sequences, (b) a second DNA sequenceencoding a substance that negatively regulates transcription,translation or secretion of the polypeptides, (c) a third DNA sequenceencoding a protease capable of hydrolyzing any of the first polypeptidesunder the conditions, and (d) one or more fourth DNA sequences encodinga second polypeptide in the second biosynthetic pathway of a secondmetabolite wherein the biosynthetic pathway and the second biosyntheticpathway involve the production of the same intermediate and the secondpolypeptide catalyzes a step after the production of the intermediate;and

[0122] (ii) the mutant cell produces more of the metabolite than theparent cell when both cells are cultivated under the conditions; and

[0123] (B) recovering the metabolite.

[0124] The present invention also relates to methods of producing ametabolite, comprising

[0125] (A) cultivating a mutant cell under conditions conducive forproduction of the metabolite, wherein

[0126] (i) the mutant cell is related to a parent cell, which comprisesone or more first DNA sequences encoding first polypeptides in thebiosynthetic pathway of the metabolite, by the introduction of a nucleicacid construct into the genome of the parent cell at a locus which isnot within (a) the first DNA sequences, (b) a second DNA sequenceencoding a protein that negatively regulates transcription, translationor secretion of the polypeptides, and (c) one or more third DNAsequences encoding a second polypeptide in the second biosyntheticpathway of a second metabolite wherein the biosynthetic pathway and thesecond biosynthetic pathway involve the production of the sameintermediate and the second polypeptide catalyzes a step prior to theproduction of the intermediate; and

[0127] (ii) the mutant cell produces less of the metabolite than theparent cell when both cells are cultivated under the conditions; and

[0128] (B) recovering the metabolite.

[0129] The present invention also relates to methods of producing afirst polypeptide, comprising

[0130] (a) forming a mutant cell by introducing a nucleic acid constructinto the genome of the parent cell at a locus which is not within thefirst DNA sequence, a second DNA sequence encoding a protein thatnegatively regulates transcription, translation or secretion of a secondpolypeptide, and a third DNA sequence encoding a protease capable ofhydrolyzing the polypeptide under conditions conducive to the productionof the first polypeptide;

[0131] (b) isolating the mutant cell which produces more of thepolypeptide than the parent cell when both cells are cultivated underthe conditions;

[0132] (c) identifying the locus wherein the nucleic acid construct hasbeen integrated;

[0133] (d) producing a cell in which a corresponding locus has beendisrupted;

[0134] (e) culturing the cell under the conditions conducive; and

[0135] (f) recovering the first polypeptide.

[0136] A corresponding locus is defined herein as a locus which encodesa polypeptide with has the same function as the polypeptide encoded bythe rescued locus.

[0137] The present invention also relates to methods of producing apolypeptide, comprising

[0138] (a) cultivating a mutant cell under conditions conducive forproduction of the polypeptide, wherein

[0139] (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence and a second DNA sequenceencoding a protease capable of hydrolyzing the polypeptide under theconditions, wherein the introduction of the nucleic acid constructspecifically enhances transcription, translation or secretion of thepolypeptide; and

[0140] (ii) the mutant cell produces more of the polypeptide than theparent cell when both cells are cultivated under the conditions; and

[0141] (b) recovering the polypeptide.

[0142] “Specific” enhancement of transcription, translation, orsecretion as used herein refers to an enhancement of one or more aspectsof the biogenesis and production of the polypeptide that is limited tothe polypeptide of interest and is not accompanied by a global effect onother polypeptides in the cell. Preferably, specific enhancement affectsonly a small number of polypeptides, including the polypeptide ofinterest. Most preferably, specific enhancement affects only thepolypeptide of interest.

[0143] Global enhancement of these biogenetic processes can bedistinguished from specific enhancement using conventional methods thatare well known in the art. For example, biosynthetic pulse-labellingwith ³H-uridine (followed by quantitation of total radioactivityincorporated into RNA) can be used to determine that a mutant cell doesnot generally synthesize mRNA at a higher or lower rate than the parentcell to which it is related. Similarly, pulse-labelling with, e.g.,³⁵S-methionine (followed by quantitation of total radioactivityincorporated into protein) can be used to determine that a mutant celldoes not generally synthesize proteins at a higher or lower rate thanthe parent cell to which it is relate. General rates of secretion can becompared between mutant and parent cells by pulse-chase labelling usingradioactive amino acids or sugars followed by quantitation ofextracellular vs. intracellular radioactivity.

[0144] These methods can also be used to determine if transcription,translation, or secretion of a limited number of other polypeptidesmight also be affected in the mutant cell. For example, capture ofspecific radiolabelled RNA transcripts by hybridization to animmobilized oligonucleotide probe can be used to assess transcriptionrates of individual genes. For translation and secretion, resolution ofradiolabelled nascent proteins by, e.g., SDS-PAGE (with or withoutimmunoprecipitation of individual proteins) can be used to compareinstantaneous rates of translation and/or secretion of individualproteins.

[0145] Polypeptides

[0146] The term “polypeptide” encompasses peptides, oligopeptides, andproteins and, therefore, is not limited to a specific length of theencoded product. The polypeptide may e native to the cell or may be aheterologous polypeptide. Preferably, it is a heterologous polypeptide.The polypeptide may also be a recombinant polypeptide which is apolypeptide native to a cell, which is encoded by a nucleic acidsequence which comprises one or more control sequences foreign to thegene. The polypeptide may be a wild-type polypeptide or a variantthereof. The polypeptide may also be a hybrid polypeptide which containsa combination of partial or complete polypeptide sequences obtained fromat least two different polypeptides where one or more of thepolypeptides may be heterologous to the cell. Polypeptides furtherinclude naturally occurring allelic and engineered variations of theabove mentioned polypeptides.

[0147] In a preferred embodiment, the polypeptide is an antibody orportions thereof.

[0148] In a preferred embodiment, the polypeptide is an antigen.

[0149] In a preferred embodiment, the polypeptide is a clotting factor.

[0150] In a preferred embodiment, the polypeptide is an enzyme.

[0151] In a preferred embodiment, the polypeptide is a hormone or ahormone variant.

[0152] In a preferred embodiment, the polypeptide is a receptor orportions thereof.

[0153] In a preferred embodiment, the polypeptide is a regulatoryprotein.

[0154] In a preferred embodiment, the polypeptide is a structuralprotein.

[0155] In a preferred embodiment, the polypeptide is a reporter.

[0156] In a preferred embodiment, the polypeptide is a transportprotein.

[0157] In a more preferred embodiment, the polypeptide is anoxidoreductase,

[0158] In a more preferred embodiment, the polypeptide is a transferase.

[0159] In a more preferred embodiment, the polypeptide is a hydrolase.

[0160] In a more preferred embodiment, the polypeptide is a lyase.

[0161] In a more preferred embodiment, the polypeptide is an isomerase.

[0162] In a more preferred embodiment, the polypeptide is a ligase.

[0163] In an even more preferred embodiment, the polypeptide is anaminopeptidase

[0164] In an even more preferred embodiment, the polypeptide is anamylase.

[0165] In an even more preferred embodiment, the polypeptide is acarbohydrase.

[0166] In an even more preferred embodiment, the polypeptide is acarboxypeptidase.

[0167] In an even more preferred embodiment, the polypeptide is acatalase.

[0168] In an even more preferred embodiment, the polypeptide is acellulase.

[0169] In an even more preferred embodiment, the polypeptide is achitinase.

[0170] In an even more preferred embodiment, the polypeptide is acutinase.

[0171] In an even more preferred embodiment, the polypeptide is adeoxyribonuclease.

[0172] In an even more preferred embodiment, the polypeptide is adextranase.

[0173] In an even more preferred embodiment, the polypeptide is anesterase.

[0174] In an even more preferred embodiment, the polypeptide is analpha-galactosidase.

[0175] In an even more preferred embodiment, the polypeptide is abeta-galactosidase.

[0176] In an even more preferred embodiment, the polypeptide is aglucoamylase.

[0177] In an even more preferred embodiment, the polypeptide is analpha-glucosidase.

[0178] In an even more preferred embodiment, the polypeptide is abeta-glucosidase.

[0179] In an even more preferred embodiment, the polypeptide is ahaloperoxidase.

[0180] In an even more preferred embodiment, the polypeptide is aninvertase.

[0181] In an even more preferred embodiment, the polypeptide is alaccase.

[0182] In an even more preferred embodiment, the polypeptide is alipase.

[0183] In an even more preferred embodiment, the polypeptide is amannosidase.

[0184] In an even more preferred embodiment, the polypeptide is amutanase.

[0185] In an even more preferred embodiment, the polypeptide is anoxidase.

[0186] In an even more preferred embodiment, the polypeptide is apectinolytic enzyme.

[0187] In an even more preferred embodiment, the polypeptide is aperoxidase.

[0188] In an even more preferred embodiment, the polypeptide is aphytase.

[0189] In an even more preferred embodiment, the polypeptide is apolyphenooxidase.

[0190] In an even more preferred embodiment, the polypeptide is aproteolytic enzyme.

[0191] In an even more preferred embodiment, the polypeptide is aribonuclease.

[0192] In an even more preferred embodiment, the polypeptide is atransglutaminase.

[0193] In an even more preferred embodiment, the polypeptide is axylanase.

[0194] In an even more preferred embodiment, the polypeptide is humaninsulin or an analog thereof.

[0195] In an even more preferred embodiment, the polypeptide is humangrowth hormone.

[0196] In an even more preferred embodiment, the polypeptide iserythropoietin.

[0197] In an even more preferred embodiment, the polypeptide isinsulinotropin.

[0198] The polypeptide also may be an enzyme involved in thebiosynthesis of a specific metabolite. The biosynthesis of a metabolitegenerally involves a biosynthetic pathway containing an array ofenzyme-catalyzed chemical reaction steps in which one or more steps maybe rate-limiting. In this embodiment of the present invention, theintegration of the nucleic acid construct into the cell's genomemodifies the production of the metabolite by modifying one or more ofthese enzyme-catalyzed steps.

[0199] The metabolite may be any organic compound of a cell which hasbeen produced by transformation of a precursor organic compound by anenzyme-catalyzed chemical reaction of the cell. The metabolite may be aprimary metabolite or a secondary metabolite. Furthermore, themetabolite may be a biosynthetic pathway intermediate or a biosyntheticpathway product. Preferably, the metabolite is an alkaloid, an aminoacid, an antibiotic, a cofactor, a drug, a fatty acid, a fungicide, aherbicide, an insecticide, an organic acid, a prosthetic group, arodenticide, a sweetener, a vitamin, a deoxysugar, a surfactant, amycotoxin, an organic acid, a sugar alcohol, a toxic metabolite, or atoxin.

[0200] Nucleic Acid Constructs

[0201] The nucleic constructs used in the methods of the presentinvention may be termed “tagged nucleic acid constructs”. “A taggednucleic acid construct” is a nucleic acid molecule containing anidentifiable nucleic acid sequence which integrates into the cell'sgenome at one or more loci thereby marking the loci. The genome is thecomplete set of DNA of a cell including chromosomal and artificialchromosomal DNA and ;extrachromosomal DNA, i.e., self-replicativegenetic elements.

[0202] The nucleic acid constructs may be any nucleic acid molecule,either single- or double-stranded, which is synthetic DNA, isolated froma naturally occurring gene, or has been modified to contain segments ofnucleic acid which are combined and juxtaposed in a manner which wouldnot otherwise exist in nature. The nucleic acid constructs may becircular or linear. Furthermore, the nucleic acid constructs may becontained in a vector, may be a restriction enzyme cleaved linearizedfragment, or may be a PCR amplified linear fragment.

[0203] The nucleic acid constructs may contain any nucleic acid sequenceof any size. In one embodiment, the nucleic acid constructs are betweenabout 10-20,000 bp in length, preferably 100-15,000 bp in length, morepreferably 500-15,000 bp in length, even more preferably 1000-15,000 bpin length, and most preferably 1,000-10,000 bp in length.

[0204] Preferably, the nucleic acid constructs have less than 40%homology, preferably less than 30% homology, more preferably less than20% homology, even more preferably less than 10% homology, and mostpreferably no homology with the locus.

[0205] Preferably, the nucleic acid constructs have less than 40%homology, preferably less than 30% identity, more preferably less than20% identity, even more preferably less than 10%, and most preferably nohomology with the DNA sequence encoding the polypeptide of interest.

[0206] The nucleic acid construct can be introduced into a cell as twoor more separate fragments. In the event two fragments are used, the twofragments share DNA sequence homology (overlap) at the 3′ end of onefragment and the 5′ end of the other. Upon introduction into a cell, thetwo fragments can undergo homologous recombination to form a singlefragment. The product fragment is then in a form suitable forrecombination with the cellular sequences. More than two fragments canbe used, designed such that they will undergo homologous recombinationwith each other to ultimately form a product suitable for recombinationwith a cellular sequence.

[0207] It will be further understood that two or more nucleic acidconstructs may be introduced into the cell as circular or linearfragments using the methods of the present invention, wherein thefragments do not contain overlapping regions as described above. It iswell known in the art that for some organisms, the introduction ofmultiple constructs into a cell results in their integration at the samelocus.

[0208] The nucleic acid constructs can contain coding or non-coding DNAsequences. Coding sequences are sequences which are capable of beingtranscribed into mRNA and translated into a polypeptide when placedunder the control of the appropriate control sequences. The boundariesof a coding sequence are generally determined by a translation startcodon ATG at the 5′-terminus and a translation stop codon at the3′-terminus. A coding sequence can include, but is not limited to, DNA,cDNA, and recombinant nucleic acid sequences.

[0209] In a preferred embodiment, the nucleic acid constructs contain aselectable marker as the identifiable nucleic acid sequence. Aselectable marker is a gene, the product of which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Integration of a selectable marker into thegenome of a host cell permits easy selection of transformed cells.Selectable marker genes for use in the methods of the present inventioninclude, but are not limited to, acetamidase (amdS), 5-aminolevulinicacid synthase (hemA), anthranilate synthase (trpC), glufosinateresistance genes, hygromycin phosphotransferase (hygB), nitratereductase (niaD), ornithine carbamoyltransferase (argB),orotidine-5′-phosphate decarboxylase (pyrG), phosphinothricinacetyltransferase (bar), and sulfate adenyltransferase (sC), as well asequivalents from other species. In a more preferred embodiment, theselectable marker is the amdS gene of Aspergillus nidulans orAspergillus oryzae, the bar gene of Streptomyces hygroscopicus, the hemAgene of Aspergillus oryzae or the pyrG gene of Aspergillus nidulans orAspergillus oryzae. Other selectable markers for use in the methods ofthe present invention are the dal genes from Bacillus subtilis orBacillus licheniformis, or markers which confer antibiotic resistancesuch as ampicillin (amp), kanamycin (kan), chloramphenicol (cam) ortetracycline resistance (tet). A frequently used mammalian marker is thedihydrofolate reductase gene (dfhr). Suitable markers for yeast hostcells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0210] In another preferred embodiment, the constructs comprise vectorsequences alone or in combination with a selectable marker, includingvector sequences containing an origin of replication, e.g., E. colivector sequences such as pUC19, pBR322, or pBluescript. For example, anE. coli vector sequence containing an origin of replication canfacilitate recovery of the construct from the host genome afterintegration due to the E. coli origin of replication. The construct canbe recovered from the host genome by digestion of the genomic DNA with arestriction endonuclease followed by ligation of the recovered constructand transformation of E. coli.

[0211] In a preferred embodiment, the nucleic acid constructs do notcontain the coding sequence of the DNA sequence for the polypeptide orportions thereof. In another preferred embodiment, the nucleic acidconstructs contain a sequence which is not homologous to the DNAsequence encoding the polypeptide in order to block the construct fromintegrating or disrupting the DNA sequence of interest.

[0212] In another preferred embodiment, the nucleic acid constructscontain one or more copies of the DNA sequence coding for thepolypeptide operably linked to control sequences. In this embodiment,the production of the polypeptide will be modified by both geneinactivation and the introduction of one or more copies of the DNAsequence.

[0213] In another preferred embodiment, the nucleic acid constructs donot contain transposable elements, i.e., transposons. A transposon is adiscrete piece of DNA which can insert itself into many different sitesin other DNA sequences within the same cell. The proteins necessary forthe transposition process are encoded within the transposon. A copy ofthe transposon may be retained at the original site after transposition.The ends of a transposon are usually identical but in inverseorientation with respect to one another.

[0214] In another preferred embodiment, the nucleic acid constructs maycontain one or more control sequences, e.g., a promoter alone or incombination with a selectable marker, wherein the control sequences uponintegration are not operably linked to the DNA sequence encoding thepolypeptide of interest. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the production of a primary RNA transcript.Such control sequences are a promoter, a signal sequence, a propeptidesequence, a transcription terminator, a polyadenylation sequence, anenhancer sequence, an attenuator sequence, and an intron splice sitesequence. Each control sequence may be native or foreign to the cell orto the polypeptide-coding sequence.

[0215] The presence of a strong promoter in the nucleic acid constructallows for additional genetic effects in addition to gene inactivationvia insertion into a structural gene (or functional transcriptionalpromoter or mRNA termination regions). The promoter may insert upstreamof a structural gene so as to enhance its transcription. Alternatively,if the promoter sequences insert in reverse gene orientation so as togenerate antisense RNA, there is the possibility of gene inactivation indiploid or higher ploidy cells. By the same mechanism, insertion of thepromoter sequences in reverse orientation may result in inactivation ofmultiple gene family encoded gene product activities.

[0216] In another preferred embodiment, the nucleic acid constructscontain a control sequence other than a promoter.

[0217] In another preferred embodiment, the nucleic acid constructs donot contain control sequences.

[0218] Locus

[0219] In the methods of the present invention, the nucleic acidconstructs are introduced at a “locus not within the DNA sequence ofinterest” or a “locus not within DNA sequences encoding polypeptides inthe biosynthetic pathway of a metabolite” which means that the nucleicacid construct is not introduced into the polypeptide-coding sequence,the control sequences thereof, and any intron sequences within thecoding sequence.

[0220] Control sequences include all components which are operablylinked to the DNA sequence and involved in the expression of thepolypeptide-coding sequence. Such control sequences are a promoter, asignal sequence, a propeptide sequence, a transcription terminator, apolyadenylation sequence, an enhancer sequence, an attenuator sequence,and an intron splice site sequence. Each of the control sequences may benative or foreign to the coding sequence.

[0221] The promoter sequence contains transcriptional control sequenceswhich mediate the expression of the polypeptide. The promoter may be anypromoter sequence including mutant, truncated, and hybrid promoters.

[0222] The signal peptide coding region codes for an amino acid sequencelinked to the amino terminus of the polypeptide which can direct theexpressed polypeptide into the cell's secretory pathway.

[0223] The propeptide coding region codes for an amino acid sequencepositioned at the amino terminus of the polypeptide. The resultantpolypeptide is known as a proenzyme or propolypeptide (or a zymogen insome cases). A propolypeptide is generally inactive and can be convertedto mature active polypeptide by catalytic or autocatalytic cleavage ofthe propeptide from the propolypeptide.

[0224] The terminator is a sequence operably linked to the 3′ terminusof the polypeptide coding sequence, and is recognized by the cell toterminate transcription of the polypeptide coding sequence.

[0225] The polyadenylation sequence is a sequence which is operablylinked to the 3′ terminus of the DNA sequence and which, whentranscribed, is recognized by the cell as a signal to add polyadenosineresidues to the transcribed mRNA.

[0226] The enhancer sequence is a sequence which can increasetranscription from a gene when located up to several kilobases from thegene. The enhancer sequencer is usually upstream of the gene.

[0227] The attenuator sequence is a sequence which regulates theexpression of a gene by determining whether the mRNA molecule containingits transcript will be completed or not.

[0228] The intron sequence is a sequence of a gene which is notrepresented in the protein product of the gene. Intron sequences aretranscribed into RNA and must be excised and the RNA molecule religatedthrough a process called intron splicing before it can be translated.

[0229] The locus may be noncontiguous or contiguous with the above-notedsequences. Preferably the locus is noncontiguous. The locus may be onthe same chromosome or the same extrachromosomal element or on adifferent chromosome or a different extrachromosomal element as that ofthe DNA sequence of interest. Furthermore, the locus may be native orforeign to the cell.

[0230] In a preferred embodiment, the locus is at least 1,000 bp, morepreferably at least 2,000 bp, and even more preferably at least 3,000bp, even more preferably at least 4,000 bp, even more preferably atleast 5,000 bp, and most preferably at least 10,000 bp from the 5′ or 3′terminus of the DNA sequence of interest.

[0231] In another preferred embodiment, the locus is on a differentchromosome than the DNA sequence encoding the polypeptide of interest.

[0232] In various methods of the present invention, the nucleic acidconstructs are introduced at a locus not within a DNA sequence encodinga protease capable of hydrolyzing the polypeptide under physiologicalconditions, which means that the nucleic acid construct is notintroduced into the protease-coding sequence, the control sequencesthereof, any intron sequences within the coding sequence, and any DNAsequences encoding proteins that positively regulate transcription,translation or secretion of the protease.

[0233] In another preferred embodiment, the locus encodes a polypeptidedifferent from the polypeptide encoded by the DNA sequence.

[0234] In another preferred embodiment, the locus encodes a glucosetransporter. Preferably, the locus has at least 60% homology, morepreferably at least 70% homoloy, even more preferably at least 80%homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:9.

[0235] In another preferred embodiment, the locus encodes amannitol-1-phosphate dehydrogenase. Preferably, the locus has at least60% homology, more preferably at least 70% homoloy, even more preferablyat least 80% homology, even more preferably at least 90% homology, andmost preferably at least 95% homology with the nucleic acid sequence ofSEQ ID NO:25.

[0236] In another preferred embodiment, the locus encodes a chitinsynthase. Preferably, the locus has at least 60% homology, morepreferably at least 70% homoloy, even more preferably at least 80%homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:56.

[0237] In another preferred embodiment, the locus encodes a heat shockprotein. Preferably, the locus has at least 60% homology, morepreferably at least 70% homoloy, even more preferably at least 80%homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:50.

[0238] In another preferred embodiment, the locus encodes a manganesesuperoxide dismutase. Preferably, the locus has at least 60% homology,more preferably at least 70% homoloy, even more preferably at least 80%homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:29.

[0239] In another preferred embodiment, the locus is a gene required foractivation of pacC, preferably a palB gene. Preferably, the locus has atleast 60% homology, more preferably at least 70% homoloy, even morepreferably at least 80% homology, even more preferably at least 90%homology, and most preferably at least 95% homology with the nucleicacid sequence of SEQ ID NO:16.

[0240] In another preferred embodiment, the locus has at least 60%homology, more preferably at least 70% homoloy, even more preferably atleast 80% homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:34.

[0241] In another preferred embodiment, the locus has at least 60%homology, more preferably at least 70% homoloy, even more preferably atleast 80% homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:39.

[0242] In another preferred embodiment, the locus has at least 60%homology, more preferably at least 70% homoloy, even more preferably atleast 80% homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:63.

[0243] In another preferred embodiment, the locus has at least 60%homology, more preferably at least 70% homoloy, even more preferably atleast 80% homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:66.

[0244] In another preferred embodiment, the locus has at least 60%homology, more preferably at least 70% homoloy, even more preferably atleast 80% homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:71.

[0245] In another preferred embodiment, the locus has at least 60%homology, more preferably at least 70% homoloy, even more preferably atleast 80% homology, even more preferably at least 90% homology, and mostpreferably at least 95% homology with the nucleic acid sequence of SEQID NO:76.

[0246] In another preferred embodiment, the locus encodes anaminopeptidase.

[0247] In another preferred embodiment, the locus encodes an amylase.

[0248] In another preferred embodiment, the locus encodes acarbohydrase.

[0249] In another preferred embodiment, the locus encodes acarboxypeptidase.

[0250] In another preferred embodiment, the locus encodes a catalase.

[0251] In another preferred embodiment, the locus encodes a catalase.

[0252] In another preferred embodiment, the locus encodes a cellulase.

[0253] In another preferred embodiment, the locus encodes a chitinase.

[0254] In another preferred embodiment, the locus encodes a cutinase.

[0255] In another preferred embodiment, the locus encodes adeoxyribonuclease.

[0256] In another preferred embodiment, the locus encodes a dextranase.

[0257] In another preferred embodiment, the locus encodes an esterase.

[0258] In another preferred embodiment, the locus encodes analpha-galactosidase.

[0259] In another preferred embodiment, the locus encodes abeta-galactosidase.

[0260] In another preferred embodiment, the locus encodes aglucoamylase.

[0261] In another preferred embodiment, the locus encodes a nalpha-glucosidase.

[0262] In another preferred embodiment, the locus encodes abeta-galactosidase.

[0263] In another preferred embodiment, the locus encodes aglucoamylase.

[0264] In another preferred embodiment, the locus encodes analpha-glucosidase.

[0265] In another preferred embodiment, the locus encodes abeta-glucosidase.

[0266] In another preferred embodiment, the locus encodes ahaloperoxidase.

[0267] In another preferred embodiment, the locus encodes an invertase.

[0268] In another preferred embodiment, the locus encodes a laccase.

[0269] In another preferred embodiment, the locus encodes a lipase.

[0270] In another preferred embodiment, the locus encodes a mannosidase.

[0271] In another preferred embodiment, the locus encodes a mutanase.

[0272] In another preferred embodiment, the locus encodes an oxidase.

[0273] In another preferred embodiment, the locus encodes a pectinolyticenzyme.

[0274] In another preferred embodiment, the locus encodes a peroxidase.

[0275] In another preferred embodiment, the locus encodes a phytase.

[0276] In another preferred embodiment, the locus encodes apolyphenoloxidase.

[0277] In another preferred embodiment, the locus encodes a proteolyticenzyme.

[0278] In another preferred embodiment, the locus encodes aribonuclease.

[0279] In another preferred embodiment, the locus encodes atransglutaminase.

[0280] In another preferred embodiment, the locus encodes a xylanase.

[0281] In a more preferred embodiment, the locus is the sequencecontained in pDSY109.

[0282] In a more preferred embodiment, the locus is the sequencecontained in pDSY112.

[0283] In a more preferred embodiment, the locus is the sequencecontained in pDSY138.

[0284] In a more preferred embodiment, the locus is the sequencecontained in pDSY141.

[0285] In a more preferred embodiment, the locus is the sequencecontained in pDSY162.

[0286] In a more preferred embodiment, the locus is the sequencecontained in pMT1936.

[0287] In a more preferred embodiment, the locus is the sequencecontained in pSMO1204.

[0288] In a more preferred embodiment, the locus is the sequencecontained in pSMOH603.

[0289] In a more preferred embodiment, the locus is the sequence of SEQID NO:9.

[0290] In a more preferred embodiment, the locus is the sequence of SEQID NO:16.

[0291] In a more preferred embodiment, the locus is the sequence of SEQID NO:25.

[0292] In a more preferred embodiment, the locus is the sequence of SEQID NO:29.

[0293] In a more preferred embodiment, the locus is the sequence of SEQID NO:34.

[0294] In a more preferred embodiment, the locus is the sequence of SEQID NO:39.

[0295] In another more preferred embodiment, the locus is the sequencecontained in p4-8. 1.

[0296] In another more preferred embodiment, the locus is the sequencecontained in p7-14.1.

[0297] In another more preferred embodiment, the locus is the sequencecontained in pHB220.

[0298] In another more preferred embodiment, the locus is the sequencecontained in pSMO717.

[0299] In another more preferred embodiment, the locus is the sequencecontained in pSMO321.

[0300] In another more preferred embodiment, the locus is the sequencecontained in pHowB571.

[0301] In another more preferred embodiment, the locus is the sequencecontained in pSMO810.

[0302] In another more preferred embodiment, the locus is the sequenceof SEQ ID NO:50.

[0303] In another more preferred embodiment, the locus is the sequenceof SEQ ID NO:56.

[0304] In another more preferred embodiment, the locus is the sequenceof SEQ ID NO:63.

[0305] In another more preferred embodiment, the locus is the sequenceof SEQ ID NO:66.

[0306] In another more preferred embodiment, the locus is the sequenceof SEQ ID NO:71.

[0307] In another more preferred embodiment, the locus is the sequenceof SEQ ID NO:76.

[0308] In another preferred embodiment, the locus does not encode atrans factor of the DNA sequence of interest. A “trans factor” is afactor which is encoded by a gene separate from the DNA sequence ofinterest which activates or represses transcription of the DNA sequence.In a more preferred embodiment, the locus does not encode a repressor ofthe DNA sequence of interest. In a more preferred embodiment, the locusdoes not encode an activator of the DNA sequence of interest.

[0309] Cells

[0310] The methods of the present invention may be used with any cellcontaining a DNA sequence encoding a polypeptide of interest includingprokaryotic cells such as bacteria, or eukaryotic cells such asmammalian, insect, plant, and fungal cells. The DNA sequence may benative or foreign to the cell. The cell may be a unicellularmicroorganism or a non-unicellular microorganism. Furthermore, the cellmay be wild-type or a mutant cell. For example, the mutant cell may be acell which has undergone classical mutagenesis or genetic manipulation.

[0311] Useful prokaryotic cells are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothernophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces urinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial cell is a Bacillus lentus, Bacilluslicheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell.

[0312] In a preferred embodiment, the cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra). Representative groupsof Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium),Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts.Examples of Basidiomycota include mushrooms, rusts, and smuts.Representative groups of Chytridiomycota include, e.g., Allomyces,Blastocladiella, Coelomomyces, and aquatic fungi. Representative groupsof Oomycota include, e.g., Saprolegniomycetous aquatic fungi (watermolds) such as Achlya. Examples of mitosporic fungi include Alternaria,Aspergillus, Candida, and Penicillium. Representative groups ofZygomycota include, e.g., Mucor and Rhizopus.

[0313] In a preferred embodiment, the fungal cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into thefamilies Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae, andSaccharomycoideae (e.g., genera Kluyveromyces, Pichia, andSaccharomyces). The basidiosporogenous yeasts include the generaFilobasidiella, Filobasidium, Leucosporidim, Rhodosporidium, andSporidiobolus. Yeast belonging to the Fungi Imperfecti are divided intotwo families, Sporobolomycetaceae (e.g., genera Bullera andSorobolomyces) and Cryptococcaceae (e.g., genus Candida). Since theclassification of yeast may change in the future, for the purposes ofthis invention, yeast shall be defined as described in Biology andActivities of Yeast (Skinner et al., 1980, Soc. App. Bacteriol.Symposium Series No. 9, 1980. The biology of yeast and manipulation ofyeast genetics are well known in the art (see, e.g., Biochemistry andGenetics of Yeast, (Bacil, M., Horecker, B.J., and Stopani, A. O. M.,editors), 2nd edition, 1987; The Yeasts (Rose, A. H., and Harrison, J.S., editors), 2nd edition, 1987; and The Molecular Biology of the YeastSaccharomyces, Strathern et al., editors, 1981).

[0314] In a more preferred embodiment, the yeast cell is a cell of aspecies of Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia.

[0315] In a most preferred embodiment, the yeast cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredembodiment, the yeast cell is a Kluyveromyces lactis cell. In anothermost preferred embodiment, the yeast cell is a Yarrowia lipolytica cell.

[0316] In another preferred embodiment, the fungal cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative. In a morepreferred embodiment, the filamentous fungal cell is a cell of a speciesof, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola,Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia,Tolypocladium, and Trichoderma.

[0317] In an even more preferred embodiment, the filamentous fungal cellis an Aspergillus cell. In another even more preferred embodiment, thefilamentous fungal cell is an Acremonium cell. In another even morepreferred embodiment, the filamentous fungal cell is a Fusarium cell. Inanother even more preferred embodiment, the filamentous fungal cell is aHumicola cell. In another even more preferred embodiment, thefilamentous fungal cell is a Mucor cell. In another even more preferredembodiment, the filamentous fungal cell is a Myceliophthora cell. Inanother even more preferred embodiment, the filamentous fungal cell is aNeurospora cell. In another even more preferred embodiment, thefilamentous fungal cell is a Penicillium cell. In another even morepreferred embodiment, the filamentous fungal cell is a Thielavia cell.In another even more preferred embodiment, the filamentous fungal cellis a Tolypocladium cell. In another even more preferred embodiment, thefilamentous fungal cell is a Trichoderma cell.

[0318] In a most preferred embodiment, the filamentous fungal cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum cell. In a most preferred embodiment, the filamentousfungal cell is a Fusarium venenatum cell (Nirenberg sp. nov.). Inanother most preferred embodiment, the filamentous fungal cell is aHumicola insolens cell or a Humicola lanuginosa cell. In another mostpreferred embodiment, the filamentous fungal cell is a Mucor mieheicell. In another most preferred embodiment, the filamentous fungal cellis a Myceliophthora thermophila cell. In another most preferredembodiment, the filamentous fungal cell is a Neurospora crassa cell. Inanother most preferred embodiment, the filamentous fungal cell is aPenicillium purpurogenum cell. In another most preferred embodiment, thefilamentous fungal cell is a Thielavia terrestris cell. In another mostpreferred embodiment, the filamentous fungal cell is a Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

[0319] Useful mammalian cells include Chinese hamster ovary (CHO) cells,HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number ofimmortalized cells available, e.g., from the American Type CultureCollection.

[0320] Introduction of Nucleic Acid Constructs into Cells

[0321] The nucleic acid construct(s) may be introduced into a cell by avariety of physical or chemical methods known in the art including, butnot limited to, transfection or transduction, electroporation,microinjection, microprojectile bombardment, alkali salts, orprotoplast-mediated transformation.

[0322] The introduction of the nucleic acid construct into a cell forinsertional mutagenesis is referred to as “DNA-tagged mutagenesis”.“DNA-tagged mutagenesis” is defined herein as the introduction of anucleic acid molecule into a cell, which leads to one or more insertionsof the nucleic acid molecule into one or more loci of the genome of thecell thereby marking the loci into which the nucleic acid molecule isinserted. The mutant cell produced by DNA-tagged mutagenesis is called atagged mutant.

[0323] Suitable procedures for transformation of Aspergillus cells aredescribed in EP 238 023 and Yelton et al, 1984, Proceedings of theNational Academy of Sciences USA 81: 1470-1474. A suitable method oftransforming Fusarium species is described by Malardier et al., 1989,Gene 78: 147-156 or in WO 96/00787. Yeast may be transformed using theprocedures described by Becker and Guarente, In Guide to Yeast Geneticsand Molecular Biology, Methods of Enzymology 194: 182-187; Ito et al.,1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

[0324] The transformation of a bacterial cell may, for instance, beaccomplished by protoplast transformation (see, e.g., Chang and Cohen,1979, Molecular General Genetics 168: 111-115), by using competent cells(see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), by electroporation (see, e.g., Shigekawa andDower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g.,Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0325] Mammalian cells may be transformed by direct uptake using thecalcium phosphate precipitation method of Graham and Van der Eb, 1978,Virology 52: 546. Other processes, e.g., electroporation, known to theart, may be used.

[0326] When the nucleic acid construct is a vector, integration into thecell's genome occurs randomly by homologous and/or non-homologousrecombination depending on the cell of choice.

[0327] In a preferred embodiment, the nucleic acid construct isintroduced into the parent cell by restriction enzyme-mediatedintegration (REMI). REMI, which is described in Schiestl and Petes,1991, Proceedings of the National Academy of Sciences USA 88: 7585-7589,is the introduction of plasmid DNA digested with a restriction enzymealong with the restriction enzyme into a cell which subsequently leadsto integration of the plasmid DNA into the genome often at a sitespecified by the restriction enzyme added. The advantage of REMIDNA-tagged mutagenesis is it can generate mutations whose molecularbasis can be easily identified.

[0328] When the nucleic acid construct is a restriction enzyme cleavedlinear DNA fragment, insertion of the construct into the cell's genomethrough REMI in the presence of the appropriate restriction enzyme israndom by virtue of the randomness of the restriction sites present inthe genome. The nucleic acid construct may insert into the cell's genomeas a single copy or as multiple copies at a single locus or at adifferent locus or at different loci. It is preferable that the nucleicacid construct insert as a single copy to facilitate the identificationand recovery of the tagged locus.

[0329] Screening of Mutant Cells

[0330] The present invention also relates to mutant cells which produce,express, synthesize or secrete more of a polypeptide or metabolite thanthe parent cell when both cells are cultivated under the conditions.

[0331] The present invention also relates to mutant cells which produce,express, synthesize or secrete more of a polypeptide or metabolite thanthe parent cell when both cells are cultivated under the conditions.

[0332] Following the introduction of a nucleic acid construct into acell, the next step is to isolate the mutant cell with the modifiedproduction of a polypeptide from a population of presumptive mutantcells. The isolation of the mutant cell preferably relies on measurementof the production of the polypeptide or the metabolite by the mutantcell relative to the parent cell when the mutant cell and the parentcell are cultured under the same conditions.

[0333] The phrase “modified production of a polypeptide” includes analteration or change of a step in the production of a polypeptide or ametabolite by the mutant cell relative to the parent cell. Such stepsinclude, but are not limited to, transcription, post-transcriptionalmodification, translation, post-translational modification, secretion,fermentation, proteolysis, down-stream processing, recovery, andpurification.

[0334] The mutant cell may be a mutant cell, for example, with improvedproduction of a specific polypeptide or metabolite or a mutant cellwhich is no longer capable or has a diminished capability of producing aspecific polypeptide or metabolite. Furthermore, the mutant cell may bea mutant cell having an increased uptake of an inorganic cofactor.

[0335] The mutant cell may also have a more desirable phenotype than theparent cell which modifies the production of a polypeptide or ametabolite. The term “phenotype” is defined herein as an observable oroutward characteristic of a cell determined by its genotype andmodulated by its environment. Such a mutant cell having a desiredphenotype includes, but is not limited to, a morphological mutant cell,a secretion mutant cell, an auxotrophic mutant cell, a conditionalmutant, a mutant cell exhibiting an altered growth rate under desiredconditions relative to the parent cell, a mutant cell resulting in therelief of overexpression mediated growth inhibition, or a mutant cellable to tolerate low oxygen conditions.

[0336] Furthermore, the mutant cell may be characterized as being amutant cell exhibiting altered production of a transcriptional activatorof a promoter or a cryptic intron-splicing-deficient mutant cell.

[0337] The isolation of a mutant cell may involve screening methodsknown in the art specific to the desired phenotype and/or thepolypeptide or the metabolite of interest. In general where a desiredphenotype is involved, a method specific to the desired phenotype may beused initially to identify the mutant cell, but then may be followed bya method specific to the polypeptide or the metabolite.

[0338] The population of presumptive mutants obtained by introducing anucleic acid construct into the cells of an organism to produce a mutantcell are first purified using standard plating techniques such as thoseused in classical mutagenesis (see, for example, Lawrence, C. W., 1991,In Christine Guthrie and Gerald R. Fink, editors, Methods in Enzymology,Volume 194, pages 273-281, Academic Press, Inc., San Diego), singlespore isolation, or enrichment techniques. The standard platingtechniques are preferably conducted in combination with a means ofdetecting the desired phenotype and/or the polypeptide or themetabolite. Different enrichment techniques may be used for increasingthe percentage of mutant cells in comparison to their wild-type orparent equivalents such as (1) direct selection which utilizes growthconditions that greatly favor the growth of the mutant; (2)counterselection, which makes use of conditions that kill the parentcells; (3) physical selection, which involves unique properties of themutant cells that enable them to be physically separated from theirparent cells; and (4) direct measurements of the amount of desiredsubstances. However, whether or not a means for identifying the mutantcell with respect to the desired phenotype and/or the polypeptide or themetabolite of interest can be incorporated into the plating medium, thepurified presumptive mutants may require further characterization toconfirm the identity of the mutant. Examples of the methods used tofurther characterize and confirm the identity of the mutant areillustrated below.

[0339] A mutant with improved production of a specific polypeptide or aspecific metabolite may be identified by using a detection method knownin the art that is specific for the polypeptide or the metabolite.Detection methods for polypeptides may include, but are not limited to,use of specific antibodies, enzymatic activity by measuring formation ofan enzyme product or disappearance of an enzyme substrate, clearingzones on agar plates containing an enzyme substrate, and biologicalactivity assays. Detection methods for metabolites may include, but arenot limited to, thin layer chromatography, high performance liquidchromatography, gas chromatography, mass spectroscopy, biologicalactivity assays, bioassays, and fluorescent activating cell sorting.

[0340] In a preferred embodiment, the specifically desired mutant cellis a mutant cell with improved production of a specific polypeptide.

[0341] In another preferred embodiment, the specifically desired mutantis a mutant with improved production of a specific metabolite; morepreferably an alkaloid, an amino acid, an antibiotic, a cofactor, adrug, a fatty acid, a fungicide, a herbicide, an insecticide, an organicacid, a pigment, a plastic precursor, a polyester precursor, aprosthetic group, a rodenticide, a sweetner, or a vitamin; and mostpreferably citric acid or lactic acid.

[0342] A prosthetic group or an organic cofactor which is a constituentof a polypeptide and/or required for biological activity may beoverproduced by isolating a mutant according to the methods of thepresent invention. Such a mutant would be particularly important wherebiosynthesis of the prosthetic group or the cofactor is a rate-limitingevent in the production of a polypeptide in a biologically active form,e.g., a hemoprotein containing heme including, but not limited to, acytochrome, specifically cytochrome P450, cytochrome b, cytochrome c₁,or cytochrome c; a globin, specifically, hemoglobin or myoglobin; anoxidoreductase, specifically a catalase, an oxidase, an oxygenase, ahaloperoxidase, or a peroxidase; or any other polypeptide containing aheme as a prosthetic group.

[0343] In a more preferred embodiment, the specifically desired mutantcell is a mutant cell overproducing an adenosine phosphate,S-adenosyl-L-methionine, biocytin, biotin, coenzyme A, coenzyme Q(ubiquinone), 5′-deoxyadenosylcobalamine, a ferredoxin, a flavincoenzyme, heme, lipoic acid, a nucleoside diphosphate, a nicotinamideadenine dinucleotide, a nicotinamide adenine dinucleotide phosphate,phosphoadenosine, phosphosulfate, pyridoxal phosphate, tetrahydrofolicacid, thiamine pyrophosphate, or a thioredoxin.

[0344] In another preferred embodiment, the specifically desired mutantcell is a mutant cell characterized with an increased uptake of aninorganic cofactor. The uptake by a cell of an inorganic cofactor whichis a constituent of a polypeptide and/or required for biologicalactivity may be increased by isolating a mutant according to the methodsof the present invention. Such a mutant would be particularly importantwhere uptake of the inorganic cofactor is a rate-limiting event in theproduction of a polypeptide in a biologically active form. In a morepreferred embodiment, the specifically desired mutant cell is a mutantcell characterized with an increased uptake of Co²⁺, Cu^(2+,) Fe²⁺,Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Mo, Ni²⁺, Se, or Zn²⁺.

[0345] In a preferred embodiment, the polypeptide or the metabolite isproduced by the mutant cell in an amount which is at least 20% greater,preferably at least 50%, more preferably at least 75%, more preferablyat least 100%, more preferably at least 100%-1000%, even more preferablyat least 200%-1000%, and most preferably at least 500%-1000% or moregreater than the cell.

[0346] In another preferred embodiment, the specifically desired mutantcell is a mutant cell which is no longer capable or has a diminishedcapability of producing a specific polypeptide. A mutant cell which isno longer capable or has a diminished capability of producing a specificpolypeptide may be identified using the same methods described above forpolypeptides, but where no or diminished production is measured relativeto the parent cell.

[0347] In a more preferred embodiment, the specifically desired mutantcell is a mutant cell which is no longer capable or has a diminishedcapability of producing a polypeptide.

[0348] In another preferred embodiment, the polypeptide is produced bythe mutant cell in an amount which is at least 20%, more preferably atleast 50%, even more preferably at least 75%, and most preferably 100%lower than the cell.

[0349] In another preferred embodiment, the specifically desired mutantcell is a mutant cell which is no longer capable or has a diminishedcapability of producing a specific metabolite. A mutant cell which is nolonger capable or has a diminished capability of producing a specificmetabolite may be identified using the same or similar methods describedabove for metabolites, but where no or diminished production is measuredrelative to the parent cell.

[0350] In a more preferred embodiment, the specifically desired mutantcell is a mutant cell which is no longer capable or has a diminishedcapability of producing a deoxysugar, a surfactant, a mycotoxin, anorganic acid, a sugar alcohol, a toxic metabolite, or a toxin; and mostpreferably an aflatoxin, beta-exotoxin, cyclopiazonic acid, an enniatin,a fusarin, kanosamine, mannitol, oxalic acid, surfactin, a tricothecene,a zearalenol, or a zearalenone.

[0351] In another preferred embodiment, the metabolite is produced bythe mutant cell in an amount which is at least 20% lower than the cell,more preferably 50%, even more preferably 75%, and most preferably 100%lower than the cell.

[0352] In another preferred embodiment, the mutant cell is amorphological mutant cell. A “morphological mutant cell” is definedherein as a mutant cell which has a desired morphology. A morphologicalmutant cell may be identified, for example, by using standard platingtechniques employing a growth medium which elicits the desiredmorphology relative to the parent cell, by microscopic examination, orby sorting vegetatively growing cells by fluorescence activated cellsorting. Such morphological mutants include, but are not limited to, amutant characterized as having superior Theological properties, e.g., ahighly-branched fungal mutant, a restricted colonial fungal mutant, or ahighly-branched restricted colonial fungal mutant which possesses rapidgrowth and low viscosity growth characteristics; a mutant whichpossesses a filamentous form during fermentation in contrast to a pelletform; a mutant which is less “sticky” preventing the colonization offermentor surfaces; a mutant with a predictable viscosity during thecourse of a fermentation; a color mutant which aids in monitoring andmaintaining the purity of a culture and high production of a polypeptideby the culture; a wettable cell which lacks, for example, a cell wall orstructural hydrophobic protein, e.g., hydrophobin; an osmoticstress-insensitive mutant which improves growth of a cell; adesiccation-insensitive mutant which improves growth of a cell; anon-spore-forming mutant which enhances the production of a polypeptide;and a non-slime-producing mutant with low viscosity growth.

[0353] Preferably, the morphological mutant cell is a color mutant, awettable mutant cell, a mutant characterized as having superiorTheological properties, an osmotic stress-insensitive mutant, adesiccation-insensitive mutant, a non-spore-forming mutant, or anon-slime-producing mutant, and most preferably a highly-branched fungalmutant, a restricted colonial fungal mutant, or a highly-branchedrestricted colonial fungal mutant.

[0354] In another preferred embodiment, the mutant cell is a secretionmutant cell. A “secretion mutant cell” is defined herein as a mutantcell which produces higher yields of one or more secreted proteins. Asecretion mutant cell may be identified by using a detection methodknown in the art that is specific for the polypeptide and comparing theyield to one or more known secreted polypeptides at the same time.Detection methods for polypeptides may include, but are not limited to,use of specific antibodies, enzymatic activity by measuring formation ofan enzyme product or disappearance of an enzyme substrate, clearingzones on agar plates containing an enzyme substrate, biological activityassays, and fluorescent activating cell sorting.

[0355] In another preferred embodiment, the specifically desired mutantcell is an auxotrophic mutant cell. An “auxotrophic mutant cell” isdefined herein as a mutant cell which has lost its ability to synthesizeone or more essential metabolites or to metabolize one or moremetabolites which modifies the production of a polypeptide by the mutantcell. An auxotrophic mutant cell may be identified using standardplating techniques by growing the presumptive mutant both in the absenceand presence of an essential metabolite. The auxotrophic mutant will notgrow in the absence of the essential metabolite. The auxotrophic mutantcan be advantageously used to selectively screen for a mutant producinga specific polypeptide of interest.

[0356] In a more preferred embodiment, the specifically desired mutantcell is an auxotrophic mutant cell unable to metabolize or synthesizeone or more of an amino acid, a fatty acid, an organic acid, apyrimidine, a purine, or a sugar; and more preferably 5-aminolevulinicacid, biotin, glucose, lactose, or maltose.

[0357] In another preferred embodiment, the specifically desired mutantcell is a conditional mutant cell. A “conditional mutant cell” isdefined herein as a mutant cell which contains one or more mutationswhose phenotypes are only observed under certain conditions and modifiesthe production of a polypeptide or a metabolite by the mutant cell.Conditional mutations can occur in virtually all genes, including thosethat control the steps in macromolecular synthesis, modification, andassembly into supermolecular structures. A conditional mutant cell maybe identified using standard plating techniques by growing thepresumptive mutant both under permissive and restrictive conditions. Forexample, a mutant strain which does not produce undesirable proteolyticactivity under nitrogen limited conditions would be desirable comparedto the parent strain which produces proteolytic activity under nitrogenlimited conditions. An additional example is an alkaline pH sensitivemutant that does not grow at alkaline pH, but may have increased ordecreased production of a desired polypeptide. A further example is amutant which is unable to grow under specifc growth conditions.

[0358] In a more preferred embodiment, the conditional mutant cell is atemperature-sensitive, acid pH sensitive, alkaline pH sensitive,antibiotic-resistant, antibiotic-sensitive, toxin-resistant,toxin-sensitive, virus-resistant, or paraquat-sensitive cell; and mostpreferably an alkaline pH sensitive mutant cell.

[0359] In another preferred embodiment, the specifically desired mutantcell is a mutant cell exhibiting an altered growth rate relative to theparent cell. A “mutant cell exhibiting an altered growth rate” isdefined herein as a mutant cell which has a doubling time that isdifferent than that of the parent cell. Such a mutant cell may beidentified by comparing the growth of the mutant cell and the parentcell under controlled fermentation conditions. Such a mutant cell mayhave improved fermentation characteristics like a shorter fermentationtime to increase productivity, or a longer fermentation time to providecontrol of the oxygen demand of a culture.

[0360] In another preferred embodiment, the specifically desired mutantcell is a mutant cell resulting in the relief of overexpression mediatedgrowth inhibition. A “mutant cell resulting in the relief ofoverexpression mediated growth inhibition” is defined herein as a mutantcell whose growth is not inhibited by the overproduction of a desiredpolypeptide or metabolite when grown under conditions that induce highlevel production of the polypeptide or the metabolite. Such a mutant maybe identified by standard plating techniques on plates with an inducingcarbon source, e.g., maltose. Mutants would be able to grow well on theinducing carbon source while the parent cells would grow poorly. Such amutant would be useful since it is known in some cells thatoverexpression of a polypeptide is toxic to the cells.

[0361] In another preferred embodiment, the specifically desired mutantcell is a mutant cell able to tolerate low oxygen conditions. A “mutantcell able to tolerate low oxygen conditions” is defined herein as amutant cell which is able to grow and produce a desired polypeptide ormetabolite under growth conditions where the dissolved oxygenconcentration is low. Such a mutant cell is particularly advantageousfor fermentations where the productivity of high cell densitiesdecreases due to oxygen transfer. A low oxygen tolerant mutant ispreferably detected by growing the mutant cell relative to the parentcell on a solid or in a liquid medium in the presence of low levels ofoxygen.

[0362] In a more preferred embodiment, the specifically desired mutantcell is a mutant cell able to tolerate low oxygen conditions in therange of about 0 to about 50% saturation, preferably about 0 to about40% saturation, even more preferably about 0% to about 30% saturation,more preferably about 0% to about 20% saturation, most preferably about0% to about 10% saturation, and even most preferably about 0% to about5% saturation.

[0363] In another preferred embodiment, the specifically desired mutantcell is a signal transduction pathway mutant cell. A “signaltransduction pathway mutant cell” is defined herein as a mutant cellwith a mutation in one or more of the genes of the pathway whichmodifies the production of a polypeptide encoded by a DNA sequence ofinterest. The term “signal transduction pathway” is defined herein as acascade of genes encoding polypeptides that are all required for theactivation or deactivation of another single polypeptide. The pathwaysenses a signal and through the cascade of genes, the signal istransduced and leads to the activation or deactivation of one or morepolypeptides. Such a mutant is preferably detected using a method whichis specific to the desired phenotype which modifies the production of apolypeptide of interest.

[0364] In a more preferred embodiment, the signal transduction pathwaymutant cell is a glucose transport signal transduction pathway mutant ora pH signal transduction pathway mutant, even more preferably a mutantin which gene required for activation of pacC has been disrupted, andmost preferably a gluT gene mutant or a palB gene mutant.

[0365] In another preferred embodiment, the specifically desired mutantcell is a mutant cell exhibiting altered production of a transcriptionalactivator of a promoter. A “mutant cell exhibiting altered production ofa transcriptional activator of a promoter” is defined herein as a mutantcell with a mutation in a gene encoding a transcriptional activatorwhich ‘turns-up’ or ‘turns-down’ a promoter of a DNA sequence encoding apolypeptide of interest.

[0366] Examples of such promoters in a bacterial cell are promoters ofthe genes of the Bacillus amyloliquefaciens alpha-amylase gene (amyQ),the Bacillus licheniformis alpha-amylase gene (amyL), the Bacilluslicheniformis penicillinase gene (penP), the Bacillus stearothermophilusmaltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene(sacB), the Bacillus subtilis xylA and xylB genes, the E. coli lacoperon, the Streptomyces coelicolor agarase gene (dagA), and theprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75:3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80:21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242:74-94; and in J. Sambrook, E. F. Fritsch, and T.Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.

[0367] Examples of such promoters in a filamentous fungal cell arepromoters of the genes encoding Aspergillus nidulans acetamidase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger neutralalpha-amylase, Aspergillus awamori or Aspergillus niger glucoamylase(glaA), Aspergillus oryzae alkaline protease, Aspergillus oryzae TAKAamylase, Aspergillus oryzae triose phosphate isomerase, Fusariumoxysporum trypsin-like protease (as described in U.S. Pat. No.4,288,627, which is incorporated herein by reference), Rhizomucor mieheiaspartic proteinase, Rhizomucor miehei lipase, and mutant, truncated,and hybrid promoters thereof. Particularly preferred promoters infilamentous fungal cells are the TAKA amylase, NA2-tpi (a hybrid of thepromoters from the genes encoding Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase), andglaA promoters.

[0368] Examples of such promoters in a yeast cell are promoters of thegenes encoding Saccharomyces cerevisiae enolase (ENO-1) gene, theSaccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase genes (ADH2/GAP), and the Saccharonyces cerevisiae3-phosphoglycerate kinase gene. Other yeast promoters are described byRomanos et al., 1992, Yeast 8:423-488.

[0369] Examples of such promoters in a mammalian cell are viralpromoters such as those of Simian Virus 40 (SV40), Rous sarcoma virus(RSV), adenovirus, and bovine papilloma virus (BPV).

[0370] In a more preferred embodiment, the mutant cell exhibits alteredproduction of a transcriptional activator of the TAKA, TAKA/NA2,Fusarium oxysporum trypsin-like protease, or a glucoamylase promoter.

[0371] In another preferred embodiment, the specifically desired mutantcell is a cryptic intron-splicing-deficient mutant cell. A “crypticintron-splicing-deficient mutant cell” is defined herein as a mutantcell which no longer recognizes and erroneously splices a cryptic intronas an authentic intron during mRNA synthesis. A crypticintron-splicing-deficient mutant cell would be particularly useful toprevent the excision or splicing of an erroneous cryptic nuclearpre-mRNA intron from a primary transcript so a biologically activesubstance is produced. In this situation, the cryptic intron is actuallypart of the coding sequence and, therefore, is not an authentic intronbut incorrectly recognized as such and erroneously spliced during mRNAsynthesis and processed by the parent cell. The introduction of a DNAsequence encoding a heterologous polypeptide into a parent cell such asa fungal host cell, particularly a filamentous fungal host cell, mayresult in this type of erroneous or aberrant splicing of the codingsequence. A cryptic intron-splicing-deficient mutant cell may beidentified by screening for increased production of a polypeptideencoded by a DNA sequence that is known to have a cryptic intron whichleads to little or no production of the polypeptide.

[0372] In another preferred embodiment, the specifically desired mutantmay be a mutant which contains two or more of the mutations describedabove.

[0373] Identification of Mutant Cells of the Present Invention

[0374] The present inventors have discovered that when certain loci in aparent cell are disrupted, the resulting mutant cell has a modifiedproduction of a polypeptide. As described above, the nucleic acidconstruct itself can have an effect on the production of a polypeptide.For example, the nucleic acid construct may comprise one or more copiesof the nucleic acid sequence encoding the polypeptide. In addition, thenucleic acid construct may comprise a promoter, transcriptionalactivators and repressors, etc.

[0375] When the nucleic acid construct itself can have an effect on theamount of polypeptide produced, expressed, synthesized or secreted, inorder to determine whether a mutant cell of the present invention hasbeen produced, one would have to rescue the locus as described below andintroduce another nucleic acid construct which does not have an effect,e.g., a selectable marker, at the same locus. If the mutant cellproduced by introducing the other nucleic acid construct at the samelocus also has an effect on the amount of polypeptide produced,expressed, synthesized or secreted, then the original mutant cell is amutant cell of the present invention.

[0376] Rescue of a Locus with the Inserted Nucleic Acid Construct andUse of a Targeting Construct

[0377] The present invention further relates to methods for rescuing alocus with the inserted nucleic acid construct comprising isolating fromthe identified mutant cell (i) the nucleic acid construct and (ii) the3′ and 5′ flanking regions of the locus of the genome where the nucleicacid construct has been integrated; and identifying the 3′ and 5′flanking regions of the locus.

[0378] The nucleic acid construct and flanking regions can be isolatedor rescued by methods well known in the art such as cleaving withrestriction enzymes and subsequent ligation and transformation of E.coli, inverse PCR, random primed gene walking PCR, or probing a libraryof the tagged mutant. The isolated nucleic acid construct with either orboth the 3′ and 5′ flanking regions is defined herein as a “targetingconstruct”.

[0379] The targeting construct includes between 100-9,000 bp, preferably200-9,000 bp, more preferably 500-7,000 bp, even more preferably1,000-7,000 bp, and most preferably 1,000-3,000 bp upstream and/ordownstream of the integration site of the nucleic acid construct.

[0380] The targeting construct of the invention may be introduced into adifferent cell to modify the production of a polypeptide similar oridentical to or completely different from the polypeptide modified inthe original cell. The other cell may be of the same or a differentspecies or of a different genera as the original cell. If the originalcell was a fungal cell, the other cell is preferably a fungal cell. Ifthe original cell was a bacterial cell, the other cell is preferably abacterial cell. If the original cell was a mammalian cell, the othercell is preferably a mammalian cell.

[0381] When the cell is a different cell, integration of the targetingconstruct preferably occurs at a target locus which is homologous to thelocus sequence of the original cell from which the targeting constructwas obtained, i.e., identical or sufficiently similar such that thetargeting sequence and cellular DNA can undergo homologous recombinationto produce the desired mutation. The sequence of the targeting constructis preferably, therefore, homologous to a preselected site of thecellular chromosomal DNA with which homologous recombination is tooccur. However, it will be understood by one of ordinary skill in theart that the likelihood of a targeting construct reinserting at a targetlocus will depend on the cell since homologous recombination frequenciesrange from almost 100% in the yeast Saccharomyces cerevisiae to as lowas 1% in Aspergillus. The targeting construct may integrate bynon-homologous recombination at a non-target locus which is not withinthe DNA sequence encoding the polypeptide of interest, but results inthe modification of the production of the polypeptide.

[0382] Preferably, the target locus includes DNA sequences that havegreater than 40% homology, preferably greater than 60% homology, morepreferably greater than 70% homology, even more preferably greater than80% homology, and most preferably greater than 90% homology with theflanking sequences of the targeting construct.

[0383] The targeting construct may contain either or both of the 3′ and5′ regions depending on whether a single cross-over or a replacement isdesired. Furthermore, the targeting construct may be modified to correctany aberrant events, such as rearrangements, repeats, deletions, orinsertions, which occurred during the introduction and integration ofthe original nucleic acid construct into the cell's genome at the locusfrom which it was originally rescued.

[0384] The targeting construct described above may be used as is, i.e.,a restriction enzyme cleaved linear nucleotide sequence, or may becircularized or inserted into a suitable vector. For example, a circularplasmid or DNA fragment preferably employs a single targeting sequence.A linear plasmid or DNA fragment preferably employs two targetingsequences. The targeting construct upon introduction into a cell, inwhich the cell comprises a DNA sequence encoding a polypeptide ofinterest, integrates into the genome of the cell at a target locus or ata nontarget locus, but preferably at a target locus, not within the DNAsequence encoding the polypeptide of interest. The target locus may beon the same chromosome or the same extrachromosomal element or on adifferent chromosome or a different extrachromosomal element as that ofthe DNA sequence of interest. The integration modifies the production ofthe polypeptide or a metabolite by the mutant cell relative to theparent cell when the mutant cell and the parent cell are cultured underthe same conditions. In a preferred embodiment, the targeting constructcontains a selectable marker.

[0385] Optionally, the targeting construct can be introduced into a cellas two or more separate fragments. In the event two fragments are used,the fragments share DNA sequence homology (overlap) at the 3′ end of onefragment and the 5′ end of the other, while one carries a firsttargeting sequence and the other carries a second targeting sequence.Upon introduction into a cell, the two fragments can undergo homologousrecombination to form a single fragment with the first and secondtargeting sequences flanking the region of overlap between the twooriginal fragments. The product fragment is then in a form suitable forhomologous recombination with the cellular target sequences. More thantwo fragments can be used, designed such that they will undergohomologous recombination with each other to ultimately form a productsuitable for homologous recombination with the cellular targetsequences.

[0386] Upon introduction of the targeting construct into a cell, thetargeting construct may be further amplified by the inclusion of anamplifiable selectable marker gene which has the property that cellscontaining amplified copies of the selectable marker gene can beselected for by culturing the cells in the presence of the appropriateselectable agent.

[0387] In a specific embodiment, the targeting construct is SphIlinearized pDSY109, HpaI linearized pDSY112, AsnI/PvuI linearizedpMT1936, NdeI linearized pDSY138, AsnI/PvuI linearized pDSY162, BglIIlinearized p4-8.1, BglII linearized p4-8.1, NarI linearized p7-14.1,BglII linearized pSMO717, BglII linearized pSMO321, NdeI linearizedpHowB571, or NdeI linearized pSMO810.

[0388] In a most preferred embodiment, the nucleic acid construct ispDSY109.

[0389] In a most preferred embodiment, the nucleic acid construct ispDSY112.

[0390] In a most preferred embodiment, the nucleic acid construct ispMT1936.

[0391] In a most preferred embodiment, the nucleic acid construct ispDSY138.

[0392] In a most preferred embodiment, the nucleic acid construct ispDSY162.

[0393] In a most preferred embodiment, the nucleic acid construct ispDSY163.

[0394] In a most preferred embodiment, the nucleic acid construct ispDSY141.

[0395] In a most preferred embodiment, the nucleic acid construct ispSMO1204.

[0396] In a most preferred embodiment, the nucleic acid construct ispSMOH603.

[0397] In a most preferred embodiment, the nucleic acid construct isp4-8.1.

[0398] In a most preferred embodiment, the nucleic acid construct isp7-14.1.

[0399] In a most preferred embodiment, the nucleic acid construct ispHB220.

[0400] In a most preferred embodiment, the nucleic acid construct ispSMO717.

[0401] In a most preferred embodiment, the nucleic acid construct ispSMO321.

[0402] In a most preferred embodiment, the nucleic acid construct ispHowB571.

[0403] In a most preferred embodiment, the nucleic acid construct ispSMO810.

[0404] In a preferred embodiment, one or more targeting constructs areintroduced into target loci. In another preferred embodiment, eachtargeting construct modifies the production of a different polypeptideor a different metabolite or a combination thereof, or results indifferent phenotypes which modify the production of differentpolypeptides or different metabolites or a combination thereof. Inanother preferred embodiment, two or more targeting constructs togetherwhen introduced into target loci act additively or synergistically tomodify the production of a polypeptide or a metabolite.

[0405] Methods of Producing a Desired Polypeptide or Metabolite fromMutant Cells

[0406] The present invention further relates to the mutant cells with adesired phenotype as host cells. Mutant cells selected for increasedproduction of a desired polypeptide or metabolite are cultivated in anutrient medium suitable for production of the polypeptide or metaboliteusing methods known in the art. For example, the cell may be cultivatedby shake flask cultivation, small-scale or large-scale fermentation(including continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermentors performed in a suitable mediumand under conditions allowing the polypeptide or metabolite to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art (see, e.g., references forbacteria and yeast; Bennett, J. W. and LaSure, L., editors, More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide or metabolite is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide or metabolite is not secreted, it isrecovered from cell lysates.

[0407] The polypeptides and metabolites may be detected using methodsknown in the art that are specific for the polypeptides and metabolitessuch as those methods described earlier or the methods described in theExamples.

[0408] The resulting polypeptide or metabolite may be recovered bymethods known in the art. For example, the polypeptide or metabolite maybe recovered from the nutrient medium by conventional proceduresincluding, but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

[0409] The polypeptides and metabolites of the present invention may bepurified by a variety of procedures known in the art including, but notlimited to, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

EXAMPLES

[0410] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use various constructs and perform the various methodsof the present invention and are not intended to limit the scope of whatthe inventors regard as their invention. Unless indicated otherwise,parts are parts by weight, temperature is in degrees centigrade, andpressure is at or near atmospheric pressure. Efforts have been made toensure accuracy with respect to numbers used (e.g., length of DNAsequences, molecular weights, amounts, particular components, etc.), butsome deviations should be accounted for.

Example 1 Strains and Materials

[0411] The starting strains were pyrG-minus Aspergillus oryzae HowB425,pyrG-minus Aspergillus oryzae HowB101, Aspergillus oryzae JaL250,Aspergillus niger strain JRoy3 (pyrGΔ), E. coli DH5α (GIBCO-BRL,Gaithersburg, Md.), and E. coli HB101 (GIBCO-BRL, Gaithersburg, Md.).

[0412] PDA plates contained 39 g/l Potato Dextrose Agar (Difco) and weresupplemented with 10 mM uridine for pyrG auxotrophs unless otherwiseindicated.

[0413] MY25 medium at pH 6.5 was composed per liter of 25 g of maltose,2.0 g of MgSO₄-7H₂O, 10 g of KH₂PO₄, 2.0 g of citric acid, 10 g of yeastextract, 2.0 g of K₂SO₄, 2.0 g of urea, and 0.5 ml of trace metalssolution. MY25 shake-flask medium was diluted 1:100 or 1:1000 with glassdistilled water for use in microtiter growth experiments (MY25/100 orMY25/1000). Cultures were grown at 34° C. 2×MY Salts pH 6.5 solution wascomposed per liter of 4 g of MgSO₄-7H₂O, 4 g of K₂SO₄, 20 g of KH₂PO₄, 4g of citric acid, 1 ml of trace metals, and 2 ml of CaCl₂-2H₂O (100 g/lstock solution.

[0414] Minimal medium transformation plates were composed per liter of 6g of NaNO₃, 0.52 g of KCl, 1.52 g of KH₂PO₄, 1 ml of trace metalssolution, 1 g of glucose, 500 mg of MgSO₄-7H₂O, 342.3 g of sucrose and20 g of Noble agar per liter (pH 6.5). Minimal medium transfer plates(pH 6.5) were composed per liter of 6 g of NaNO₃, 0.52 g of KCl, 1.52 gof KH₂PO₄, 1 ml of trace elements, 1 g of glucose, 500 mg of MgSO₄-7H₂O,and 20 g Noble agar.

[0415] The trace metals solution (1000×) was composed per liter of 22 gof ZnSO₄-7H₂O, 11 g of H₃BO₃, 5 g of MnCl₂-4H₂O, 5 g of FeSO₄-7H₂O, 1.6g of CoCl₂-5H₂O, 1.6 g of (NH₄)₆Mo₇O₂₄, and 50 g of Na₄EDTA.

[0416] COVE plates were composed per liter of 343.3 g of sucrose, 20 mlof COVE salts solution, 10 ml of 1 M acetamide, 10 ml of 3 M CsCl, and25 g of Nobel agar. The COVE salts (50×) solution was comprised of 26 gof KCl, 26 g of MgSO₄-7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE tracemetals solution. COVE trace metals solution was composed of (per liter):0.04 g of NaB₄O₇-10H₂O, 0.040 g of CuSO₄-5H₂O, 0.70 g of FeSO₄-H₂O, 0.80g of Na₂MoO₂-2H₂O, and 10 g of ZnSO₄.

[0417] YEG medium was composed per liter of 5 g yeast extract and 20 gdextrose.

[0418] CM-1 agar plates at pH 6.5 were composed per liter of 0.25 g ofNaCl, 0.5 g of MgSO₄-7H₂O, 1.9 g of K₂HPO₄, 3.6 g of KH₂PO₄, 0.1 ml oftrace metals solution, 30 g of Bacto agar (Difco), pH 6.5. 11 ml of 10%urea, and 67 ml of 30% maltose.

[0419] CD medium was composed per liter of 1 g of MgSO₄-7H₂O, 1 g ofK₂SO₄, 15 g of KH₂PO₄, 0.25 ml of trace metals solution, 0.7 g of yeastextract (Difco), 20 g of beta-cyclodextrin (Sigma C-4767). 3 ml of 50%urea, and 2 ml of 15% CaCl₂-2H₂O.

[0420] G1-gly medium was composed per liter of 18 g of yeast extract(Difco), 80 g of 75% glycerol, and 0.5 g of CaCl₂-2H₂O.

[0421] OL-1 medium (pH 7.0) was composed per liter of 15 g of KH₂PO₄, 1g of MgSO₄-7H₂O, 1 g of K₂SO₄, 0.25 ml of trace metals solution, 0.3 gof CaCl₂-2H₂O (autoclaved separately), 2 g of Difco yeast extract(Difco), 0.5 g of urea (autoclaved separately), and 10 g of glucose.

[0422] OL-6 medium (pH 7.0) was composed per liter of 15 g of KH₂PO₄, 1g of MgSO₄-7H₂O, 1 g of K₂SO₄, 0.25 ml of trace metals solution, 0.3 gof CaCl₂-2H₂O (autoclaved separately), 2 g of Difco yeast extract(Difco), 3 g of urea (autoclaved separately), and 60 g of glucose.

[0423] YPM medium was composed of 10 g of Bactopeptone and 5 g of yeastextract dissolved in 500 ml of water and autoclaved, to which 50 ml of asterilized 20% maltose solution was added.

[0424] MTBCDUY was composed per liter of 0.3 g of MgSO₄-7H₂O, 0.3 g ofK₂SO₄, 5 g of KH₂PO₄, 0.013 g of urea, 0.01 g of yeast extract, 0.1 g ofmaltose, 4.88 g of uridine, and 0.25 ml of trace metal solution 1adjusted to pH 6.5.

[0425] 4×MTBCDUY was composed per liter of 0.3 g of MgSO₄-7H₂O, 0.3 g ofK₂SO₄, 5 g of KH₂PO₄, 0.052 g of urea, 0.04 g of yeast extract, 0.4 g ofmaltose, 4.88 g of uridine, and 0.25 ml of trace metal solution 1.

[0426] MDU1B was composed per liter of 45 g of Maltodextrin MD01, 1.0 gof MgSO₄-7H₂O, 1.0 g of NaCl, 2.0 g of K₂SO₄, 12.0 g of KH₂PO₄, 7.0 g ofyeast extract, 0.5 ml of trace metal solution, and 0.1 ml of pluronicacid. The trace metal solution consisted of 13.9 g of FeSO₄-7H₂O, 8.45 gof MnSO₄-H₂O, 6.8 g of ZnCl₂, 2.5 g of CuSO₄-5H₂O, 2.5 g of NiCl₂-6H₂O,and 3 g of citric acid. The pH of the shake flask medium was adjusted to5.0 before being autoclaved.

[0427] 1/5MDU2BP was composed per liter of 9 g of maltose, 0.2 g ofMgSO₄-7H₂O, 0.4 g of K₂SO₄, 0.2 g of NaCl, 2.4 g of KH₂PO₄, 1.0 g ofurea, 1.4 g of yeast extract, and 0.1 ml of trace metal solution 1.

[0428] Trace metal solution 1 was composed per liter of 13.8 g ofFeSO₄-7H₂O, 8.5 g of MnSO₄-H₂O, 14.3 g of ZnSO₄-7H₂O, 2.5 g ofCuSO₄-5H₂O, 0.5 g of NiCl₂-6H₂O, and 3.0 g of citric acid.

[0429] YPG plates was composed per liter of 4.0 g yeast extract, 1.0 gof K₂HPO₄, 0.5 g of MgSO₄-7H₂O, 15.0 g of dextrose, and 20.0 g of agar.

Example 2 Construction of Aspergillus oryzae HowB430

[0430]Aspergillus oryzae HowB430 was constructed to contain a lipasegene from Humicola lanuginosa (LIPOLASE™ gene, Novo Nordisk A/S,Bagsvaerd, Denmark).

[0431] pBANe8 was constructed as described below to contain theTAKA/NA2-tpi leader hybrid promoter, the lipase gene from Humicolalanuginosa, the AMG terminator, and the full-length Aspergillus nidulansamdS gene as a selectable marker.

[0432] PCR was employed to insert NsiI sites flanking the full-lengthamdS gene of pToC90 (Christensen et al., 1988, Biotechnology 6:1419-1422) using primers 1 and 2 below and to insert an EcoRI site atthe 5′ end and a SwaI site at the 3′ end of the NA2-tpi leader hybridpromoter of pJaL292 (FIG. 1) using primers 3 and 4 below. The primerswere synthesized with an Applied Biosystems Model 394 DNA/RNASynthesizer (Applied Biosystems, Inc., Foster City, Calif.) according tothe manufacturer's instructions. Primer 1:5′-ATGCATCTGGAAACGCAACCCTGA-3′ (SEQ ID NO:1) Primer 2:5′-ATGCATTCTACGCCAGGACCGAGC-3′ (SEQ ID NO:2)

[0433] Amplification reactions (100 μl) were prepared usingapproximately 0.2 μg of either pToC90 or pJaL292 as the template. Eachreaction contained the following components: 0.2 μg of plasmid DNA, 48.4pmol of the forward primer, 48.4 pmol of the reverse primer, 1 mM eachof dATP, dCTP, dGTP, and dTTP, 1×Taq polymerase buffer, and 2.5 U of Taqpolymerase (Perkin-Elmer Corp., Branchburg, N.J.). The reactions wereincubated in an Ericomp Thermal Cycler programmed as follows: One cycleat 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1minute, 55° C. for 1 minute and 72° C. for 2 minutes.

[0434] The PCR products were electrophoresed on a 1% agarose gel toconfirm the presence of a 2.7 kb amdS fragment and a 0.6 kb NA2-tpifragment.

[0435] The PCR products were subsequently subcloned into pCRII using aTA Cloning Kit (Invitrogen, San Diego, Calif.) according to themanufacturer's instructions. The transformants were then screened byextracting plasmid DNA from the transformants using a QIAwell-8 PlasmidKit (Qiagen, Inc., Chatsworth, Calif.) according to the manufacturer'sinstructions, and restriction digesting the plasmid DNA with either NsiIor EcoRI/SwaI followed by agarose electrophoresis to confirm thepresence of the correct size fragments, 2.7 kb and 0.6 kb, respectively,for the NsiI amdS fragment and SwaI/EcoRI NA2-tpi fragment. In order toconfirm the PCR products, the products were sequenced with with anApplied Biosystems Model 373A Automated DNA Sequencer (AppliedBiosystems, Inc., Foster City, Calif.) on both strands using the primerwalking technique with dye-terminator chemistry (Giesecke et al., 1992,Journal of Virol. Methods 38: 47-60) using the M13 reverse (−48) and M13forward (−20) primers (New England Biolabs, Beverly, Mass.) and primersunique to the DNA being sequenced. The plasmids from the correcttransformants were then digested with the restriction enzymes for whichthe plasmids were designed, separated on a 1% agarose gel, and purifiedusing a FMC SpinBind Kit (FMC, Rockland, Me.) according to themanufacturer's instructions.

[0436] pKS6 (FIG. 2), which contains the TAKA promoter, a polylinker,the AMG terminator, and the Aspergillus nidulans pyrG gene, was digestedwith EcoRI and SwaI to remove a portion of the TAKA promoter. Thisregion was replaced with the NA2-tpi PCR product to produce pBANe13(FIG. 3).

[0437] pBANe13 was digested with NsiI to remove the Aspergillus nidulanspyrG gene. This region was then replaced with the full length amdS genePCR product described above to produce pBANe6 (FIG. 4).

[0438] PCR was used to insert SwaI and PacI flanking sites on thefull-length Humicola lanuginosa lipase gene of pMHan37 (FIG. 5) usingprimers 5 and 6 below. Primers 5 and 6 were synthesized as describedabove. Primer 5: 5′-ATTTAAATGATGAGGAGCTCCCTTGTGCTG-3′ (SEQ ID NO:5)Primer 6: 5′-TTAATTAACTAGAGTCGACCCAGCCGCGC-3′ (SEQ ID NO:6)

[0439] The amplification reaction (100 μl) contained the followingcomponents: 0.2 μg of pMHan37, 48.4 pmol of primer 5, 48.4 pmol ofprimer 6, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerasebuffer, and 2.5 U of Taq polymerase. The reaction was incubated in anEricomp Thermal Cycler programmed as follows: One cycle at 95° C. for 5minutes followed by 30 cycles each at 95° C. for 1 minute, 55° C. for 1minute, and 72° C. for 2 minutes. Two μl of the reaction waselectrophoresed on an agarose gel to confirm the amplification of thelipase gene product of approximately 900 bp.

[0440] The PCR amplified lipase gene product was then subcloned intopCRII using a TA Cloning Kit. The transformants were screened byextracting plasmid DNA from the transformants using a QIAwell-8 PlasmidKit, restriction digesting the plasmid DNA with SwaI/PacI, andsequencing the DNA according to the method described above to confirmthe PCR product.

[0441] The lipase gene was excised from the pCRII plasmid by digestingwith SwaI and PacI and subsequently subcloned into SwaI/PacI digestedpBANe6 to produce pBANe8 (FIG. 6).

[0442] pBANe8 was digested with PmeI and the linear PmeI fragmentcontaining the NA2-tpi promoter, the lipase gene from Humicolalanuginosa, and the AMG terminator was isolated by preparative agaroseelectrophoresis using 40 mM Tris-acetate-1 mM disodium EDTA (TAE)buffer.

[0443]Aspergillus oryzae HowB430 was generated by transformation ofAspergillus oryzae HowB425 with the linear PmeI fragment according tothe following procedure.

[0444]Aspergillus oryzae HowB425 was grown in 100 ml of 1% yeastextract-2% peptone-1% glucose at 32° C. for 16-18 hours with agitationat 150 rpm. The mycelia were recovered by filtration through a 0.45 μmfilter until approximately 10 ml remained on the filter, washed with 25ml of 1.0-1.2 M MgSO₄-10 mM sodium phosphate pH 6.5, filtered as before,washed again as before until 10 ml remained, and then resuspended in 10ml of 5 mg/ml NOVOZYM 234™ (Novo Nordisk A/S, Bagsvaerd, Denmark) in 1.2M MgSO₄-10 mM sodium phosphate pH 6.5 (0.45 μm filtered) in a 125 mlEhrlenmeyer flask. The suspension was incubated with gentle agitation at50 rpm for approximately one hour at 37° C. to generate protoplasts. Avolume of 10 ml of the protoplast/mycelia preparation was added to a 30ml Corex centrifuge tube, overlaid with 5 ml of 0.6 M sorbitol-10 mMTris-HCl pH 7.5, and centrifuged at 3600×g for 15 minutes in a swingingbucket rotor to recover the protoplasts. The protoplasts were recoveredfrom the buffer interface with a Pasteur pipet. The protoplasts werethen washed with five volumes of STC, centrifuged, and then rewashed andcentrifuged as before. The protoplasts were resuspended in STC to afinal concentration of 2×10⁷ protoplasts per ml.

[0445] Transformation of Aspergillus oryzae HowB425 for amdS selectionwas conducted with protoplasts at a concentration of 2×10⁷ protoplastsper ml. Ten μg of DNA were added to 100 μl of protoplasts. A volume of250 μl of PEG solution (60% PEG 4000-10 mM CaCl₂-10 mM Tris-HCl pH 8.0)was then added and the mixture was placed at 37° C. for 30 minutes.Three ml of 1 M sorbitol-10 mM CaCl₂-10 mM Tris pH 7.5 (STC) was addedand the mixture was plated on Cove plates supplemented with 10 mMuridine selecting for amdS. The plates were incubated 7-10 days at 34°C. Transformants were transferred to plates of the same medium andincubated 3-5 days at 37° C. The transformants were purified bystreaking spores and picking isolated colonies using the same plates ofthe same medium without sucrose under the same conditions.

Example 3 Construction of Aspergillus oryzae HowB427

[0446]Aspergillus oryzae HowB425 was co-transformed with pMHan37 andpSO2 (FIG. 7) to construct Aspergillus oryzae HowB427 to contain thelipase gene from Humicola lanuginosa behind the TAKA promoter.

[0447] pSO2 (FIG. 7) was constructed from a genomic library ofAspergillus oryzae 1560. The genomic library of Aspergillus oryzae 1560was constructed by first partially digesting Aspergillus oryzae 1560genomic DNA with Sau3A (New England Biolabs, Beverly, Mass.). Four unitsof Sau3A were used to digest 10 μg of Aspergillus oryzae 1560 genomicDNA using conditions recommended by the manufacturer. The reaction wascarried out at 65° C., and samples were taken at 5 minute intervals(from 0 to 50 minutes). The reactions were placed on ice and stopped bythe addition of EDTA to 10 mM. These digests were then run on a 1%agarose gel with ethidium bromide, and the region of the gel containingDNA from 3 kb to 9 kb was excised. The DNA was then purified from thegel slice using Beta-Agarase I using a protocol provided by themanufacturer (New England Biolabs, Beverly, Mass.). The size-selectedDNA was then ligated into EMBL 4 arms according to the manufacturer'sinstructions (Clontech, Palo Alto, Calif.) at 16° C. overnight usingconditions recommended by the manufacturer. The ligation reaction waspackaged and titered using a Gigapack II Packaging Kit (Stratagene, LaJolla, Calif.) according to the manufacturer's protocol. A total of16,000 recombinant plaques were obtained, and the library was amplifiedusing a protocol provided by the manufacturer.

[0448] Appropriate dilutions of the genomic library were made to obtain7000 plaques per 150 mm petri plate as described in the protocolsprovided with the EMBL 4 arms. The plaques were lifted to Hybond-N pluscircular filters (Amersham, Cleveland, Ohio) using standard protocols(Sambrook et al., 1989, supra). The filters were fixed using UVcrosslinking, and prehybridized at 42° C. (5×SSPE, 35% formamide). Thegenomic library was probed at low stringency (35% formamide, 5×SSPE at42° C.) with a 500 bp fragment consisting of the Aspergillus niger pyrGgene which was labeled with ³²P using a random prime DNA labeling kit(Boehringer Mannheim, Indianapolis, Ind.). A 3.8 kb HindIII fragment wasisolated from one phage and subcloned into a pUC118 cloning vector toproduce pSO2.

[0449] The co-transformation of Aspergillus oryzae HowB425 was conductedusing the procedure described in Example 2 except selection was onMinimal medium transformation plates. Transformants were transferred toMinimal medium transfer plates and incubated 3-5 days at 37° C. Thetransformants were then purified by streaking spores and pickingisolated colonies using the same transfer plates under the sameconditions.

Example 4 Construction of Plasmids pSO122, pDSY81, and pDSY82

[0450] pSO122 was constructed as described below to contain a 1.5 kbfragment of the Aspergillus oryzae pyrG gene.

[0451] PCR was used to generate pSO122 by introducing a BamHIrestriction site at the 5′ end of the pyrG gene of pSO2 using primers 7and 8 shown below. Primers 7 and 8 were synthesized with an AppliedBiosystems Model 394 DNA/RNA Synthesizer according to the manufacturer'sinstructions. Primer 7: 5′-GCGGGATCCCTAGAGTAGGGGGTGGTGG-3′ (SEQ ID NO:7)Primer 8: 5′-GCGGGATCCCCCCTAAGGATAGGCCCTA-3′ (SEQ ID NO:8)

[0452] The amplification reaction (50 μl) contained the followingcomponents: 2 ng of pSO2, 48.4 pmoles of the forward primer, 48.4 pmolesof the reverse primer, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taqpolymerase buffer, and 2.5 U of Taq polymerase (Perkin-Elmer Corp.,Branchburg, N.J.). The reaction was incubated in an Ericomp ThermalCycler programmed as follows: One cycle at 95° C. for 5 minutes followedby 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute and 72° C.for 2 minutes. The PCR product was isolated by electrophoresis on a 1%agarose gel.

[0453] The isolated PCR product was digested with BamHI and cloned intothe BamHI site of pBluescript SK- (Stratagene, La Jolla, Calif.) toyield pSO122 (FIG. 8). The only homology between the genome ofAspergillus oryzae HowB430 and pSO122 was in the 5′ end of the pyrGinsert since the rest of the pyrG fragment was deleted from Aspergillusoryzae HowB430 as described in Example 2.

[0454] In order to reduce the frequency of targeting to this homologousregion in the genome and since pSO122 contains two BamHI sites, twoderivatives of pSO122, pDSY81 and pDSY82 (FIG. 8), were constructed inwhich one of the BamHI sites was destroyed. The plasmids pDSY81 andpDSY82 were constructed by partially digesting pSO122 with BamHI,filling-in the 5′ overhangs with the Klenow fragment, closing down theplasmid by ligation and subsequent transformation into E. coli DH5α(Sambrook et al., 1989, supra). The transformants were then screened byextracting plasmid DNA from the transformants using a QIAwell-8 PlasmidKit and restriction digesting the plasmid DNA with BamHI to determine ifone of the BamHI sites had been destroyed. Plasmids with one of theBamHI sites destroyed were digested with NsiI/BamHI to determine whichBamHI site had been destroyed.

Example 5 Aspergillus oryzae HowB430 Transformation with pSO122, pDSY81,or pDSY82

[0455] Protoplasts of Aspergillus oryzae HowB430 were prepared asdescribed in Example A 5-15 μl aliquot of DNA (circular pSO122, pDSY81linearized with 4 to 12 U of EcoRI, or pDSY82 linearized with 15 U ofBamHI) was added to 0.1 ml of the protoplasts at a concentration of2×10⁷ protoplasts per ml in a 14 ml Falcon polypropylene tube followedby 250 μl of 60% PEG 4000-10 mM CaCl₂-10 mM Tris-HCl pH 7, gently mixed,and incubated at 37° C. for 30 minutes. The transformations were madeeither with 5 μg of circular pSO122, 6 μg of linearized pDSY81, or 6 μgof linearized pDSY82. Three ml of SPTC (1.2 M sorbitol-10 mM CaCl₂-10 mMTris pH 8) were then added and the suspension was gently mixed. Thesuspension was mixed with 12 ml of molten overlay agar (1×COVE salts, 1%NZ amine, 0.8 M sucrose, 0.6% Noble agar) or 3 ml of STC medium and thesuspension was poured onto a Minimal medium plate. The plates wereincubated at 37° C. for 3-5 days.

[0456] The transformation frequencies of the circular pSO122transformations ranged from about 100 to 200 transformants/μg. A libraryof ˜120,000 DNA-tagged transformants of Aspergillus oryzae HowB430 wasobtained.

[0457] The transformation frequencies of the EcoRI REMI pDSY81transformations ranged from about 60 to 100 per μg. An EcoRI REMIlibrary of ˜28,000 DNA-tagged transformants of Aspergillus oryzaeHowB430 was generated.

[0458] The transformation frequencies of the BamHI REMI pDSY82transformations ranged from about 80 to 110 transformants/tg. A BamHIREMI library of ˜27,000 DNA-tagged transformants of Aspergillus oryzaeHowB430 was obtained.

[0459] HindIII and SalI REMI libraries of Aspergillus oryzae HowB430were also prepared using pDSY81 as described above.

[0460] The transformation frequencies of the HindIII REMI pDSY81transformations ranged from about 80 to 120 per μg. A HindIII REMIlibrary of 35,000 DNA-tagged transformants of Aspergillus oryzae HowB430was generated.

[0461] The transformation frequencies of the SalI REMI pDSY81transformations ranged from about 80 to 120 per μg. A SalI REMI libraryof 25,000 DNA-tagged transformants of Aspergillus oryzae HowB430 wasgenerated.

[0462] The Aspergillus oryzae HowB430 tagged mutant library pools weredesignated “h” for pSO122; “e” for pDSY81 digested with EcoRI withsubsequent transformation in the presence of EcoRI; “b” for pDSY82digested with BamHI with subsequent transformation in the presence ofBamHI; “hIII” for pDSY81 digested with HindIII with subsequenttransformation in the presence of HindIII; and “s” for pDSY81 digestedwith SalI with subsequent transformation in the presence of SalI. Therewere 123 “h” pools, 28 “e” pools, 23 “b” pools, 55 “hIII” pools, and 25“s” pools.

[0463] The libraries described above were pooled into groups of ˜1000transformants and stored in 10% glycerol at −80° C.

Example 6 Characterization of Integration Events in “REMI” Aspergillusoryzae HowB430 Transformants

[0464] Genomic DNA was isolated from 26 of the EcoRI REMI transformants(“e” pool) described in Example 5 according to the following procedure.Each transformant was grown in 5 ml of YEG medium for 24 hours at 37° C.in a small Petri plate. Mycelia were then collected from each culture byfiltration through Whatman filter paper No. 1 (Whatman, SpringfieldMill, England) and transferred to a 1.7 ml centrifuge tube. The myceliapreparations were frozen in dry ice and dried in a SpeedVac (SavantInstruments, Inc., Farmingdale, N.Y.) overnight at room temperature. Thefrozen mycelia preparations were ground to a fine powder with a spearedspatula and then the ground mycelia were resuspended in 0.5 ml of lysisbuffer (100 mM EDTA, 10 mM Tris pH 8.0, 1% Triton X-100, 50 mMguanidine-HCl, 200 mM NaCl). RNase was added to each preparation to afinal concentration of 20 μg/ml, and the preparations were incubated at37° C. for 30 minutes. Protease K was then added to each preparation toa final concentration of 0.1 mg/ml, and the preparations were incubatedat 50° C. for 1 hour. The preparations were centrifuged at 13,000×g for15 minutes, and the supernatants were applied to QIAprep-8-well strips(Qiagen, Chatsworth, Calif.). The wells were washed once with 0.5 ml ofPB and 0.75 ml of PE supplied by the manufacturer (Qiagen, Chatsworth,Calif.). After removing excess PE from each well, the DNAs were elutedfrom the wells in 200 μl of TE buffer (10 mM Tris-1 mM EDTA pH 7.0).

[0465] The genomic DNA was digested with either EcoRI to determinewhether integration occurred into genomic EcoRI sites or SnaBI todetermine whether or not the integration events were random throughoutthe genome by Southern hybridization according to the proceduredescribed by Sambrook et al., 1989, supra. Southern blots of the digestswere probed with a 1.6 kb NheI pyrG fragment obtained from pSO122 (FIG.8) labeled with dioxygenin using a Genius Kit according to themanufacturer's instructions. The blot was prehybridized for 2 hours andhybridized overnight at 42° C. in DIG Easy Hyb. The blot was washed andprocessed as recommended by the manufacturer.

[0466] The Southern blot demonstrated that in 13 of 26 transformants,EcoRI linearized pDSY81 integrated into an EcoRI site in the genome, andthe distribution of the integration events appeared to be random. In 20of the 26 transformants, only a single copy of the plasmid wasintegrated while in 6 of the transformants at least 2 copies wereintegrated at the same locus. In order to determine if the bias (of 50%)towards integration at EcoRI sites was due to REMI, genomic DNA wasisolated as described above from 16 Aspergillus oryzae HowB425transformants, in which the EcoRI enzyme was heat inactivated beforetransformation with EcoRi linearized pDSY81 according to the proceduredescribed in Example 5, and submitted to Southern blot analysis asdescribed above. Southern analysis of these transformants demonstratedthat in none of the transformants did the plasmid integrate at an EcoRIsite in the genome.

Example 7 Lipase Expression Screening

[0467] The Aspergillus oryzae HowB430 tagged mutant library “h”, “e”,and “b” pools described in Example 5 were assayed for lipase expression.

[0468] For 96-well plate screens, MY25 medium was diluted 1000-foldusing a diluent made of equal volumes of sterile water and 2×MY Salts pH6.5 solution. For 24-well plate methods, MY25 medium was diluted100-fold using a diluent made of equal volumes of sterile water and 2×MYSalts pH 6.5 solution.

[0469] Primary 96-well plate screens involved the dilution of sporesfrom distinct pools into MY25/1000 so that one spore on average wasinoculated per well when 50 μl of medium was dispensed into the wells.After inoculation, the 96-well plates were grown for 7 days at 34° C.under static conditions. Cultures were then assayed for lipase activityas described below. Mutants of interest were inoculated directly into24-well plates containing MY25/100 and were grown for 7 days at 34° C.Cultures were then assayed for lipase activity as described below.Mutants of interest were then plated on COVE plates to produce spores,spread on PDA plates to produce single colonies, and then 4 singlecolonies from each isolate were tested in the 24-well plate methoddescribed above.

[0470] The lipase assay substrate was prepared by diluting 1:50 ap-nitrophenylbutyrate stock substrate (21 μl of p-nitrophenylbutyrate/mlDMSO) into MC buffer (4 mM CaCl₂-100 mM MOPS pH 7.5) immediately beforeuse. Standard lipase (LIPOLASE™, Novo Nordisk A/S, Bagsvaerd, Denmark)was prepared to contain 40 LU/ml of MC buffer containing 0.02% alphaolefin sulfonate (AOS) detergent. The standard was stored at 4° C. untiluse. Standard lipase was diluted {fraction (1/40)} in MC buffer justbefore use. Broth samples were diluted in MC buffer containing 0.02% AOSdetergent and 20 μl aliquots were dispensed to wells in 96-well platesfollowed by 200 μl of diluted substrate. Using a plate reader, theabsorbance at 405 nm was recorded as the difference of two readingstaken at approximately 1 minute intervals. Lipase units/ml (LU/ml) werecalculated relative to the lipase standard.

[0471] The results of the 96-well screen followed by the 24-well screenidentified for further evaluation 53 transformants from the pSO122transformations and 44 transformants from the pDSY81 or pDSY82 REMItransformations. These identified transformants produced higher levelsof lipase than the control strains Aspergillus oryzae HowB427 andAspergillus oryzae HowB430.

Example 8 Shake Flask and Fermentation Evaluation

[0472] The highest lipase-producing DNA-tagged mutants described inExample 7 were then plated onto COVE plates to produce spores for shakeflask and fermentation evaluations.

[0473] Shake flask evaluations were performed by inoculating 300-500 μlof a spore suspension (0.02% Tween-80 plus spores from the COVE plates)into 25 ml of MY25 medium at pH 6.5 in a 125 ml shake flask. The shakeflasks were incubated at 34° C. for 3 days at 200 rpm. Samples weretaken at day 2 and day 3 and lipase activity was measured as describedin Example 7.

[0474] The same DNA-tagged mutants were grown in a 2 liter lab fermentorcontaining medium composed of Nutriose, yeast extract, (NH₄)₂HPO₄,MgSO₄-7H₂O, citric acid, K₂SO₄, CaCl₂-H₂O, and trace metals solution at34° C., pH 7, 1000-1200 rpm for 8 days. Lipase activity was measured asdescribed in Example 7.

[0475] The results obtained are shown in Table 1 below where the lipaseyield of either Aspergillus oryzae HowB427 or Aspergillus oryzae HowB430as a control is normalized to 1.0. TABLE I Lipase Expression by DNATagged Mutants 24-well # Screened Plate Shake Flask Ferm. Strain in96-well Results Results Results Description Construction Pool Plates(LU/ml) (LU/ml) (LU/ml) HowB427 HowB425 + pMHan37 NA NA 1.2 0.6 1.0HowB430 HowB425 + pBANe8 NA NA 1.0 1.0 NA DEBY10.3 pDSY81 + BamHI b1 8081.7 2.2 3.9 DEBY203.3 pDSY81 + EcoRI e1 707 2.4 2.1 1.8 DEBY599.3pDSY81 + BamHI b18 443 1.5 2.4 4.1 DEBY932 pDSY81 + EcoRI e21 1092 1.91.9 3.6 DEBY1058 pDSY81 + BamHI b22 80 2.3 2.4 3.8 DEBY1204.3.3 pDSY81 +EcoRI e26 1260 1.9 2.0 3.0 HINL603 pDSY81 + HindIII hi3-7 NA 2.8 2.0 3.3HowL91.1 pSO122 h32 1134 1.9 2.3 3.3 HowL214.2 pSO122 h9 861 1.9 2.3 3.0HowL301.4 pSO122 h58 731 2.3 2.8 3.6 HowL371.3 pSO122 h92 592 1.8 2.53.5 HowL442.1 pSO122 h7 1095 2.3 2.6 4.5 HowL465.2 pSO122 h8 1003 2.32.2 3.2 HowL500.1 pSO122 h99 885 2.3 2.7 3.4 HowL554.1 pSO122 h120 8921.9 2.3 3.6 HowL795.4 pSO122 h29 1029 3.0 3.8 4.9

[0476] As shown in Table I, the mutants produced approximately 2- to4-fold more lipase than the control strain Aspergillus oryzae HowB427and approximately 3- to 6-fold more lipase than the control strainAspergillus oryzae HowB430 when grown in shake flasks. The mutants alsoproduced approximately 2- to 5-fold more lipase than the control strainAspergillus oryzae HowB427 when grown in fermentors.

Example 9 Rescue of Plasmid DNA and Flanking DNA from High lipaseExpressing Mutants

[0477] The plasmid DNA (pSO122, pDSY81, or pDSY82) and genomic flankingloci were isolated from mutants Aspergillus oryzae DEBY10.3, DEBY599.3,DEBY932, DEBY1058, DEBY1204.3.3, and HIN603.

[0478] Genomic DNA was isolated from mutants Aspergillus oryzaeDEBY10.3, DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and HIN603according to the following procedure. Spore stocks of each mutant wereinoculated into 150 ml of YEG medium and were grown overnight at 37° C.and 250 rpm. The mycelia were harvested from each culture by filtrationthrough Miracloth (Calbiochem, La Jolla, Calif.) and rinsed twice withTE. The mycelia preparations were then frozen quickly in liquid nitrogenand ground to a fine powder with a mortar and pestle. The powderedmycelia preparations were each transferred to a 50 ml tube and 20 ml oflysis buffer was added. RNAse was added to each preparation to a finalconcentration of 20 μg/ml, and the preparations was incubated at 37° C.for 30 minutes. Protease K was then added to each preparation to a finalconcentration of 0.1 mg/ml, and the preparations were incubated at 50°C. for 1 hour. The preparations were then centrifuged at 15,000×g for 20minutes to pellet the insoluble material. Each supernatant was appliedto a Qiagen MAXI column (Qiagen, Chatsworth, Calif.) which wasequilibrated with QBT provided by the manufacturer. The columns werethen washed with 30 ml of QC provided by the manufacturer. DNA waseluted from each column with 15 ml of QF provided by the manufacturerand then recovered by precipitation with a 0.7 volume of isopropanol andcentrifugation at 15,000×g for 20 minutes. The pellets were finallywashed with 5 μl of 70% ethanol, air-dried, and dissolved in 200 μl ofTE.

[0479] Two μg aliquots from each of the Aspergillus oryzae DEBY10.3,DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and HIN603 genomic DNApreparations were digested separately with BglII HpaI, NarI, NdeI, SphI,and StuI. The restriction endonucleases did not cut pDSY82 which allowedthe isolation of the integrated plasmid and the flanking genomic DNA.The digested genomic DNAs were then ligated in a 20 μl reaction with T4DNA ligase.

[0480] The ligated DNA preparations were each transformed into E. coliHB101 or E. coli DH5α. The transformants were then screened byextracting plasmid DNA from the transformants, restriction digesting theinserts to confirm they are derived from pDSY82, and sequencing theinserts according to the method described above using primers specificto pDSY82.

[0481] Transformant E. coli HB101-pDSY112 contained the HpaI rescuedlocus from mutant Aspergillus oryzae DEBY599.3. Transformant E. coliHB101-pDSY109 contained the SphI rescued locus from mutant Aspergillusoryzae DEBY10.3. Transformant E. coli HB101-pDSY138 contained the NdeIrescued locus from mutant DEBY932. Transformant E. coli HB101-pDSY141contained the BglII rescued locus from mutant DEBY1058. Transformant E.coli DH5α-pSMO1204 contained the BglII rescued locus from mutantAspergillus oryzae DEBY1204.3.3. Transformant E. coli DH5α-pSMOH603contained the BglII rescued locus from mutant Aspergillus oryzae HIN603.

Example 10 Characterization of Aspergillus oryzae DEBY599.3 RescuedLocus pDSY112

[0482] The Aspergillus oryzae DEBY599.3 rescued locus pDSY112 containing1625 bp was sequenced according to the method described in Example 2.The nucleic acid sequence (SEQ ID NO:9) and the deduced amino acidsequence (SEQ ID NO:10) are shown in FIG. 9. The nucleic acid sequencesuggested that integration occurred within the promoter of a glucosetransporter about 150 bp upstream of the ATG start codon. The openreading frame was punctuated by an intron. The predicted protein (SEQ IDNO:10) shared 31.6% and 24.8% identity with the glucose transportersfrom yeast (SEQ ID NO:11) and human (SEQ ID NO:12), respectively, and20.1% identity with an inositol transporter from yeast (SEQ ID NO:13).Glucose transporters have very distinct predicted secondary structureswith 12 membrane spanning domains. Kyte-Doolittle plots of theAspergillus oryzae DEBY599.3 rescued locus predicted 12 membranespanning domains similar to the yeast and human glucose transporters.

[0483] In order to confirm that the rescued flanking DNA was the genedisrupted in Aspergillus oryzae DEBY599.3, a Southern blot ofAspergillus oryzae HowB101 and Aspergillus oryzae DEBY599.3 genomic DNApreparations digested with BglII was prepared and analyzed according tothe procedure described in Example 6. The blot was probed with theAspergillus oryzae DEBY599.3 rescued flanking DNA at 42° C. in DIG EasyHyb. The blot was then washed and processed using protocols providedwith a Genius Kit.

[0484] A BglII band of 2.7 kb from Aspergillus oryzae HowB101 hybridizedwith the probe, while an ˜8 kb BglII band from Aspergillus oryzaeDEBY599.3 hybridized to the probe. The size difference corresponded tothe length of the plasmid integrated during REMI confirming the DNArescued from Aspergillus oryzae DEBY599.3 was flanking the insertion.

Example 11 Aspergillus oryzae Transformation with HpaI LinearizedpDSY112 and Lipase Expression Screening

[0485]Aspergillus oryzae HowB430 was transformed with HpaI digestedpDSY112 and the transformants were recovered using the methods describedin Example 5. Totally, 216 transformants were grown in 24 wellmicrotiter plates in {fraction (1/100)} strength MY25 medium. Sampleswere taken at 4 and 6 days and assayed for lipase activity as describedin Example 7. An equal number of low, average and high producing lipasetransformants were spore purified and retested in 24 well microtitercultures as described above. These purified transformants were alsotested in shake flasks in full-strength MY25 medium as described inExample 8. The top five producing transformants were then grown in a 2liter fermentor as described in Example 8. Lipase activity was measuredas described in Example 7.

[0486] The results obtained are shown in Table 2 below where the lipaseyield of Aspergillus oryzae HowB430 is normalized to 1.0. TABLE 2 StrainFermentation Results (Relative LU/ml) HowB430 1.0 DEBY599.3 1.7112T90.2.2 2.3 112T100.4.2 2.1 112T344.2.1 1.7 112T142.2 2.0 112T59.22.4

[0487] All five retransformants produced approximately the same level oflipase activity as the original tagged strain Aspergillus oryzaeDEBY599.3 when grown under fermentation conditions. In order todetermine if the pDSY112 had integrated at the same homologous locus inthe genome, a Southern blot of Aspergillus oryzae HowB430, Aspergillusoryzae DEBY599.3 and the pDSY112 transformants genomic DNA preparationsdigested with BglII was prepared and analyzed according to the proceduredescribed in Example 6. The blot was probed with the Aspergillus oryzaeDEBY599.3 rescued flanking DNA at 42° C. in DIG Easy Hyb. The blot waswashed and processed using protocols provided with a Genius Kit.

[0488] A BglII band of 2.7 kb from Aspergillus oryzae HowB430 hybridizedwith the probe, while an ˜8 kb BgllI band from Aspergillus oryzaeDEBY599.3 hybridized to the probe. A wild-type BglII band of 2.7 kb anda second band corresponding to the transforming DNA hybridized to theprobe in all of the transformants. Therefore, none of theretransformants had exact gene replacements.

Example 12 Characterization of Aspergillus oryzae DEBY10.3 Rescued LocuspDSY109

[0489] The 3.4 and 2.2 kb regions on either side of the integrationevent of the Aspergillus oryzae DEBY10.3 rescued locus pDSY109 weresequenced according to the procedure described in Example 2. The nucleicacid sequence suggested that the integration event occurred within theopen reading frame of a palB gene. palB genes encode a cysteine proteaseinvolved in the signal transduction pathway that signals ambient pH.

[0490] The genomic library of Aspergillus oryzae HowB430 was constructedby first partially digesting Aspergillus oryzae HowB430 genomic DNA withTsp509I. Four units of Tsp509 were used to digest 3.5 μg of Aspergillusoryzae HowB430 genomic DNA using conditions recommended by themanufacturer. The reaction was carried out at 65° C., and samples weretaken at 5 minute intervals (from 0 to 50 minutes). The reactions wereplaced on ice and stopped by the addition of EDTA to 10 mM. Thesedigests were then run on a 1% agarose gel with ethidium bromide, and theregion of the gel containing DNA from 3 kb to 9 kb was excised. The DNAwas then purified from the gel slice using Beta-Agarase I using aprotocol provided by the manufacturer (New England Biolabs, Beverly,Mass.). The size-selected DNA was then ligated into Lambda ZipLox EcoRIarms according to the manufacturer's instructions at 16° C. overnightusing conditions recommended by the manufacturer. The ligation reactionwas packaged and titered using a Gigapack GoldIII Packaging Kitaccording to the manufacturer's protocol. 8×10⁶ recombinant plaques wereobtained, and the library was amplified using a protocol provided by themanufacturer.

[0491] The genomic library was screened to obtain a genomic clone ofpalB. Appropriate dilutions of the genomic library were made to obtain7000 plaques per 150 mm petri plate as described in the protocolsprovided with the Lambda ZipLox arms. The plaques were lifted toHybond-N plus circular filters using standard protocols (Sambrook etal., 1989, supra). The filters were fixed using UV crosslinking, andprehybridized at 42° C. in DIG Easy Hyb. The filters were hybridizedwith a DIG-labeled 0.25 kb palB probe. The probe was labeled withdioxygenin using a Genius Kit and PCR amplified with the followingprimers synthesized with an Applied Biosystems Model 394 DNA/RNASynthesizer according to the manufacturer's instructions:5′-CTGCCGTCGAAGGTGTCCAAG-3′ (SEQ ID NO:14) 5′-ATTGTGGCCCCTATGTGGATT-3′(SEQ ID NO:15)

[0492] The parameters for PCR are as described in Example 2. The filterswere washed and processed post-hybridization using protocols providedwith the Genius Kit. Several positive plaques were identified andpurified to homogeneity using standard protocols (Sambrook et al., 1989,supra).

[0493] The nucleotide sequence was determined for the palB geneaccording to the method described in Example 2. The nucleic acidsequence (SEQ ID NO:16) and the deduced amino acid sequence (SEQ IDNO:17) are shown in FIG. 10. The open reading frame was interrupted by 3introns. The Aspergillus oryzae PalB protein (SEQ ID NO:17) shared 66.4%identity with the Aspergillus nidulans PalB protein (SEQ ID NO:18). Thesite of insertion also contained a highly conserved domain of 37 aminoacids (SEQ ID NO:19) similar to that derived from the Neurospora crassaNADH dehydrogenase (SEQ ID NO:20) which was probably a piece ofmitochondrial DNA that inserted during transformation or rescue in E.coli.

[0494] A Southern blot of Aspergillus oryzae DEBY10.3 and Aspergillusoryzae HowB101 genomic DNA digested with BglII was prepared according tothe emthod described in Example 6. The blot was probed with theAspergillus oryzae DEBY10.3 rescued flanking DNA to confirm that therescued flanking DNA was the gene disrupted in Aspergillus oryzaeDEBY10.3.

[0495] A BglII band of ˜7.5 kb from Aspergillus oryzae HowB101hybridized to the probe while a band of 12 kb from Aspergillus oryzaeDEBY10.3 hybridized to the probe. The size difference was the expectedsize for one plasmid copy being integrated confirming the locus rescuedwas disrupted in Aspergillus oryzae DEBY10.3.

[0496] Because the integration event in Aspergillus oryzae DEBY10.3would be predicted to lead to a nonfunctional PalB protein, Aspergillusoryzae DEBY10.3 was tested for growth at pH 8.0 and pH 6.5. Aspergillusnidulans palB minus strains are unable to grow at pH 8.0 but are able togrow at pH 6.5. Aspergillus oryzae HowB430 and Aspergillus oryzaeDEBY10.3 were grown in Minimal medium with 10 mM uridine at either pH8.0 or pH 6.5. As predicted, Aspergillus oryzae DEBY10.3 was unable togrow at pH 8.0.

Example 13 Construction of pMT1936

[0497] pMT1936 was constructed to contain a disruption cassette of palBusing the following primers synthesized with an Applied Biosystems Model394 DNA/RNA Synthesizer according to the manufacturer's instructions.100752: 5′-GGTTGCATGCTCTAGACTTCGTCACCTTATTA (SEQ ID NO:21) GCCC-3′100753: 5′-TTCGCGCGCATCAGTCTCGAGATCGTGTGTCG (SEQ ID NO:22) CGAGTACG-3′100754: 5′-GATCTCGAGACTAGTGCGCGCGAACAGACATC (SEQ ID NO:23) ACAGGAACC-3′100755: 5′-CAACATATGCGGCCGCGAATTCACTTCATTCC (SEQ ID NO:24) CACTGCGTGG-3′

[0498] The Aspergillus oryzae palB 5′ flanking sequence and the sequenceencoding the N-terminal part of the palB product were PCR amplified fromgenomic DNA of Aspergillus oryzae A1560 obtained according to the methoddescribed in Example 2. Approximately 0.05 μg of DNA template and 5pmole of each of the two primers 100755 and 100754 were used.Amplification was performed with the polymerase Pwo as described by themanufacturer (Boehringer Mannheim, Indianapolis, Ind.). Amplificationproceeded through 40 cycles. Part of the reaction product was phenolextracted, ethanol precipitated, digested with restriction enzymes EcoRIand XhoI and a fragment of approximately 1.05 kb was isolated by agarosegel electrophoresis.

[0499] The Aspergillus oryzae palB 3′ flanking sequence and the sequenceencoding the C-terminal part of the palB gene product were obtained asdescribed above except that primers 100753 and 100752 were used foramplification and the PCR product was digested with restriction enzymesXhoI and XbaI before gel electrophoresis to recover a fragment ofapproximately 1.50 kb.

[0500] The two digested and purified PCR fragments described above wereligated in a three part ligation with the purified 2.7 kb EcoRI-XbaIfragment from the vector pJaI400 (FIG. 11) to produce pMT1935 (FIG. 12).The palB 5′ and 3′ flanks of pMT1935 are separated by BssHII, SpeI, andXhoI sites introduced via PCR primers 100754 and 100753.

[0501] To insert an Aspergillus oryzae pyrG gene between the palB 5′flank and the 3′ flank of pMT1935, the 3.5 kb HindIII fragment ofpJaL394 (FIG. 13) containing the repeat flanked pyrG gene was clonedinto HindIII cut, dephosphorylated and purified pBluescript II SK (−).Plasmids with inserts in either orientation were obtained. One plasmid,pMT1931 (FIG. 14), was selected in which the SpeI site of thepBluescript polylinker was downstream of the pyrG gene and the XhoI sitewas upstream of the pyrG gene. The pyrG gene was isolated as a 3.5 kbSpeI-XhoI fragment and inserted in SpeI and XhoI digested and purifiedpMT1935 to produce the disruption plasmid pMT1936 (FIG. 15).

[0502] The pyrG selectable palB disruption cassette can be isolated frompMT1936 as a 6.2 kb NotI fragment (NotI cutting in polylinkers) or as a5.5 kb AseI-PvuI fragment (AseI and PvuI cutting within the actual palB5′ and 3′ flanking sequences).

Example 14 Aspergillus oryzae Transformation with AlseI/PvuI palBDisruption Cassette from pMT1936 and Lipase Screening

[0503]Aspergillus oryzae HowB430 was transformed using the sametransformation procedure described in Example 5 with a 5.5 kb AseI/PvuIfragment obtained from pMT1936. The linear fragment for transformationwas isolated by digestion of pMT1936 with AseI and PvuI and separationof the fragment on a 1% agarose gel using a QIAquick Gel Extraction Kitaccording to the manufacturer's instructions. The transformants werethen tested for growth on Minimal medium plates at pH 6.5 or pH 8.0.

[0504] The results showed that 13 of the 128 transformants testedpossessed the palB minus phenotype as indicated by the inability to growat pH 8.0. The 13 palB minus strains and 13 of the transformants thatwere able to grow at pH 8.0 were spore purified and then evaluated in24-well plate and shake flask cultures for lipase production using themethods described in Examples 7 and 8, respectively. The results areshown in Table 3 below.

[0505] Southern blots of the genomic DNA from an Aspergillus oryzae palBminus mutant, an Aspergillus oryzae palB plus strain, and Aspergillusoryzae HowB430 were performed to determine if the AsnI/PvuI transformingDNA fragment had integrated as a clean replacement into the palB locus.The genomic DNAs were prepared according to the procedure described inExample 9, digested with PvuI, and electrophoresed on a 0.8% agarosegel. The DNAs were transferred to a Hybond N⁺ membrane using 0.4 N NaOHand capillary action. The blot was UV crosslinked prior toprehybridization at 65° C. in Rapid Hyb. The blot was then probed with a0.9 kb AsnI/SpeI fragment from pMT1936. The 0.9 kb fragment was isolatedfrom an agarose gel slice using QiaQuick spin column afterelectrophoreses on a 1% agarose gel. The fragment was labeled usingVistra ECF Random Prime Labeling Kit. The blots were prehybridized andhybridized at 65° C. in Rapid Hyb (Amersham, Cleveland, Ohio), and thenwashed twice for 5 minutes in 2×SSC, 0.1% SDS at 65° C. and twice for 10minutes in 0.2×SSC, 0.1% SDS at 65° C. Following the washes, the blotwas processed for detection using the Vistra ECF Signal AmplificationKit (Amersham, Cleveland, Ohio) and the STORM860 Imaging System(Molecular Dynamics, Sunnyvale, Calif.).

[0506] The Southern blot results demonstrated that the probe hybridizedto a band of 6 kb from Aspergillus oryzae HowB430. A clean disruptionwould be expected to hybridize to about an 8 kb PvuI band. The Southernblot results further showed that some of the palB minus strains hadclean disruptions while others did not. The Southern blot results aresummarized in Table 3.

[0507] Three of the palB minus strains were also run under fermentationconditions according to the procedure described in Example 8. Theresults obtained are shown in Table 3 below where the lipase yield ofAspergillus oryzae HowB430 is normalized to 1.0. The three palB minusstrains performed better or close to the same as the original taggedmutant Aspergillus oryzae DEBY10.3. TABLE 3 Fermentation PalB 24 wellShake flasks results Southern Strain phenotype LU/ml LU/ml LU/ml patternHowB430 plus 1.0 1.0 1.0 wild type palB3-1 plus 1.2 1.1 1.7 wild-typeand other palB4-1 plus 1.0 0.8 1.4 wild-type and other palB5-1 minus 1.41.4 2.0 disrupted palB8-1 plus 0.9 1.0 NA wild-type and other palB18-1plus 0.9 NA NA wild-type and other palB27-1 plus 1.0 NA NA wild-type andother palB29-1 minus 0.8 NA NA other palB30-1 plus 0.9 NA 1.0 wild-typeand other palB31-1 minus 1.3 NA NA other palB37-1 plus 0.8 NA NAwild-type and other palB39-1 plus 0.9 NA NA wild-type and other palB41-1plus 1.0 0.8 NA wild-type and other palB42-1 plus 1.2 1.0 NA wild-typeand other palB43-1 minus 1.3 1.2 NA other palB69-1 plus 1.2 1.4 NAwild-type and other palB71-1 minus 1.2 1.3 1.8 other palB72-1 minus 1.51.6 2.0 other palB75-1 minus 1.3 1.4 1.3 other palB76-1 minus 1.6 1.32.0 clean disruption palB79-1 plus 1.2 1.0 NA wild-type and other

Example 15 Characterization of Aspergillus oryzae DEBY932 Rescued LocuspDSY138

[0508] The Aspergillus oryzae DEBY932 rescued locus pDSY138 containing1625 bp was sequenced according to the method described in Example 2.The nucleic acid sequence (SEQ ID NO:25) and deduced amino acid sequence(SEQ ID NO:26) are shown in FIG. 16. The nucleic acid sequence showedthat the EcoRI site of the REMI integration was 810 bp upstream of theATG start codon for an open reading frame and the deduced amino acidsequence (SEQ ID NO:26) had significant identity to mannitol-1-phosphatedehydrogenases from E. coli and Bacillus subtilis. The open readingframe coded for a predicted protein of 319 amino acids, and shared 13.3%and 34.7% identity with the E. coli (SEQ ID NO:27) and the Bacillussubtilis (SEQ ID NO: 28) mannitol-1-phosphate dehydrogenases,respectively.

[0509] A Southern blot of Aspergillus oryzae DEBY932 and Aspergillusoryzae HowB430 genomic DNA preparations digested with NdeI was preparedand analyzed according to the method described in Example 14. The blotwas probed with the Aspergillus oryzae DEBY932 rescued flanking DNA toconfirm that the rescued flanking DNA is the gene disrupted in DEBY932.

[0510] An NdeI band of approximately 5 kb from Aspergillus oryzaeHowB430 hybridized to the rescued locus while a band of approximately 10kb from Aspergillus oryzae DEBY932 hybridized to the probe confirmingthat the rescued locus was the disrupted locus in Aspergillus oryzaeDEBY932.

Example 16 Aspergillus oryzae Transformation with NdeI LinearizedpDSY138 and lipase Expression Screening

[0511]Aspergillus oryzae HowB430 was transformed with NdeI digestedpDSY138 and the transformants were recovered using the methods describedin Example 5. Totally, 180 recovered transformants were grown in 24 wellmicrotiter plates in {fraction (1/100)} strength MY25, and samples weretaken at 4 and 6 days for lipase assays as described in Example 7. Thetop 11 highest lipase producing and 1 average lipase producingtransformants were spore purified and retested in 24 well microtitercultures. These purified transformants were also evaluated in shakeflasks in full-strength MY25 as described in Example 8. The top twoproducers were also grown in a 2 liter fermentor as described in Example8. Lipase activity was measured as described in Example 7.

[0512] The results obtained are shown in Table 4 below where the lipaseyield of Aspergillus oryzae HowB430 was normalized to 1.0. The top twolipase producers produced essentially the same amount of lipase activityas the original tagged mutant Aspergillus oryzae DEBY932. TABLE 4Fermentation Results Strain (Relative LU/ml) Southern Results HowB4301.0 Wild-type DEBY932.3.3 2.1 Disrupted 138T83.1.1 2.2 Disrupted138T102.1.1 1.9 Disrupted

[0513] A Southern blot of Aspergillus oryzae DEBY932, Aspergillus oryzaeHowB430 and pDSY138 genomic DNA preparations digested with NdeI wasprepared and analyzed as described in Example 14 to determine if pDSY138had integrated at the homologous locus producing gene replacements inthe transformants using the Aspergillus oryzae DEBY932 rescued flankingDNA as a probe.

[0514] The Southern blot showed that an NdeI band of approximately 5 kbfrom Aspergillus oryzae HowB430 hybridized to the rescued locus while aband of approximately 10 kb from Aspergillus oryzae DEBY932 hybridizedto the probe. In Table 4, the column labeled Southern results indicatedwhether the transformants had a wild-type NdeI fragment of the sizeobserved in the parent strain Aspergillus oryzae HowB430 or whether thetransformants had a band corresponding to the disrupted size observed inAspergillus oryzae DEBY932.

Example 17 Characterization of Aspergillus oryzae DEBY1058 Rescued LocuspDSY141

[0515] The Aspergillus oryzae DEBY1058 rescued locus pDSY141 containingapproximately 1 kb was sequenced according to the method described inExample 2. The nucleic acid sequence demonstrated that the rescued locuscontained flanking DNA from only one side of the BamHI REMI integrationevent, and the pDSY141 sequence had rearranged.

[0516] A Southern blot of Aspergillus oryzae DEBY1058 genomic DNAdigested with BamHI as probed with the Aspergillus oryzae DEBY1058rescued flanking DNA was prepared and analyzed as described in Example14 to confirm that the rescued flanking DNA is the gene disrupted inAspergillus oryzae DEBY1058.

[0517] The Southern analysis showed that the pDSY82 DNA had integratedas a REMI event at a BamHI site, but more than one copy of pDSY82 hadintegrated which suggested why the rescued plasmid had rearranged andonly contained one side of the flanking DNA.

[0518] In order to obtain the other flanking piece, a genomic clone(pDSY163) was isolated from the Aspergillus oryzae HowB430 genomiclibrary, prepared as described in Example 6, using a ³²P-labeled 0.5 kbfragment of the rescued genomic DNA from Aspergillus oryzae DEBY1058.The probe was labeled using a Prime-It Kit according to themanufacturer's instructions (Stratagene, La Jolla, Calif.). Five platesof approximately 7000 plaques each were plated, and the plaques werelifted to Hybond-N⁺ as described in Example 12. The filters wereprehybridized at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS and 200 μg/mlof sheared and denatured salmon sperm DNA for 1 hour. The denaturedprobe was added, and the filters were hybridized overnight at 42° C. Thefilters were washed in 1×SSC, 0.1% SDS for 5 minutes at 65° C. twice, in0.1×SSC, 0.1% SDS at 65° C. for 15 minutes twice, and in 2×SSC at roomtemperature for 10 minutes. The filters were exposed to X-ray film, and12 positive plaques were picked and purified using standard protocols(Sambrook et al., 1989, supra). Plasmid DNA was isolated from thepurified genomic clones using the excision protocol provided with theLambda ZipLox EcoRI Arms Kit.

[0519] The nucleotide sequence of 3.6 kb of the genomic clone wasdetermined as described in Example 2. The nucleic acid sequence (SEQ IDNO:29) and deduced amino acid sequence (SEQ ID NO:30) are shown in FIG.17. The nucleic acid sequence showed that the BamHI site of integrationin the mutant is 250 bp downstream of the stop codon for an open readingframe that encodes a protein (SEQ ID NO:30) which shared significantidentity with manganese superoxide dismutase from Saccharomycescerevisiae (SEQ ID NO:31).

[0520] Since the site of integration in Aspergillus oryzae DEBY1058 was250 bp downstream of the stop codon for the manganese superoxidedismutase gene, the effect of this integration on expression of themanganese superoxide dismutase was determined. Saccharomyces cerevisiaestrains lacking a functional manganese superoxide dismutase aresensitive to paraquat when grown in the presence of oxygen. Aspergillusoryzae DEBY1058 and Aspergillus oryzae HowB430 were grown in 24 wellmicrotiter plates in 1 ml of YEG medium supplemented with 10 mM uridineand either 0, 2, 4, 6, 8, 10 or 20 mM paraquat at 34° C. with shaking.Aspergillus oryzae HowB430 grew at concentrations of paraquat up to 8 mMwhile growth of Aspergillus oryzae DEBY1058 was inhibited by 2 mMparaquat. The data indicated that the integration event 250 bpdownstream of the stop codon for manganese superoxide dismutase inAspergillus oryzae DEBY1058 reduced expression of manganese superoxidedismutase.

Example 18 Construction of pDSY162

[0521] pDSY162 was constructed to contain a disruption cassette formanganese superoxide dismutase by PCR amplification of a 3179 bpXbaI/KpnI fragment of genomic DNA containing the manganese superoxidedismutase gene using the following primers synthesized with an AppliedBiosystems Model 394 DNA/RNA Synthesizer according to the manufacturer'sinstructions. 970738: 5′-GCTCTAGATCGTCGGAGCTCATGTCGGCGATT (SEQ ID NO:32)TTAC-3′ 970739: 5′-GCGGTACCACGCCTAGAGCAAAGTATAAATAA (SEQ ID NO:33)GGAA-3′

[0522] The amplification reaction (100 μl) contained the followingcomponents: 0.2 μg of the pDSY163, 48.4 pmol of primer 970738, 48.4 pmolof primer 979739, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taqpolymerase buffer, and 2.5 U of Taq polymerase. The reaction wasincubated in an Ericomp Thermal Cycler programmed as follows: One cycleat 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1minute, 55° C. for 1 minute, and 72° C. for 2 minutes. Two μl of thereaction were electrophoresed on an agarose gel to confirm theamplification of the PCR product of approximately 3179 bp.

[0523] The PCR product was subcloned into pCR®TOPO using a TOPO TACloning Kit (Invitrogen, San Diego, Calif.). The transformants were thenscreened by extracting plasmid DNA from the transformants using aQIAwell-8 Plasmid Kit according to the manufacturer's instructions,restriction digesting the plasmid DNA using XbaI/KpnI to confirm thepresence of the correct size fragment, and sequencing the DNA accordingto the method described in Example 2 to confirm the PCR product.

[0524] The plasmids containing the manganese superoxide dismutase insertwere digested with XbaI and KpnI and separated on a 1% agarose gel. A3.1 kb manganese superoxide dismutase fragment was purified using aQIAquick Gel Extraction Kit according to the manufacturer'sinstructions. The purified fragment was ligated with pBluescriptSK-digested with XbaI and KpnI to produce pDSY161 (FIG. 18). Theligation reaction was used to transform E. coli DH5α.

[0525] The transformants were then screened by extracting plasmid DNAfrom the transformants using a QIAwell-8 Plasmid Kit according to themanufacturer's instructions and digesting the plasmids with HindIII todetermine which clones were correct.

[0526] pDSY161 was digested with HindUII to remove a 600 bp fragment,and the digestion was electrophoresed on a 1% agarose gel. A 5.4 kbvector fragment was isolated using a QIAquick Gel Extraction Kitaccording to the manufacturer's instructions, and ligated to the 3.5 kbHindIII fragment from pJaL394 (FIG. 13) containing apyrG gene repeat toproduce pDSY162 (FIG. 19). The ligation reaction was used to transformE. coli DH5α.

[0527] The transformants were then screened by extracting plasmid DNAfrom the transformants using a QIAwell-8 Plasmid Kit according to themanufacturer's instructions and digesting them with HindIII to determinewhich plasmids contained the expected 3.5 kb HindIII fragment inpDSY162.

Example 19 Aspergillus oryzae Transformation with AsnI/PvuI ManganeseSuperoxide Dismutase Disruption Cassette and lipase Screening

[0528]Aspergillus oryzae HowB430 was transformed with a 5.8 kb AseI/PvuIfragment containing the manganese superoxide dismutase disruptioncassette using the same transformation procedure described in Example 5.The linear fragment for transformation was isolated by digestion ofpDSY162 with AseI and PvuI and separation of the fragment on a 1%agarose gel using a QIAquick Gel Extraction Kit according to themanufacturer's instructions. The transformants were then tested forgrowth on Minimal medium plates at pH 6.5.

[0529] Six transformants were obtained and were tested for sensitivityto paraquat as described in Example 17. Four of the 6 transformants wereparaquat sensitive indicative of the manganese superoxide dismutasedisruption minus phenotype although the four paraquat sensitive strainswere not equally sensitive to paraquat. As shown in Table 5 below, adash means not sensitive to paraquat, ++ means sensitive means sensitiveto intermediate levels of paraquat and +++ means inhibited by even 2 mMparaquat. All of the transformants were spore purified and tested in 24well and shake flask cultures for lipase production according to theprocedures described in Examples 7 and 8. The results tabulated in Table5 below show that following transformation of Aspergillus oryzae HowB430with the manganese superoxide dismutase disruption cassette,transformants sensitive to paraquat on average produced higher LIPOLASE™levels than Aspergillus oryzae HowB430.

[0530] Southern blots of the genomic DNA from an Aspergillus oryzaemanganese superoxide dismutase minus mutant, an Aspergillus oryzaemanganese superoxide dismutase plus strain, and Aspergillus oryzaeHowB430 were performed as described in Example 14 to determine if theAsnI/PvuI transforming DNA fragment had integrated as a cleanreplacement into the manganese superoxide dismutase locus.

[0531] The results of the Southern blot (Table 5) showed that strainssensitive to even 2 mM paraquat were disrupted at the manganesesuperoxide dismutase locus while those sensitive to intermediate levelsof paraquat have both a wild-type locus and the disrupted cassettelocus. The Souther blot and LIPOLASE™ yield results together suggeststhat expression of both full length and truncated manganese superoxidedismutase in the same cell leads to an intermediate sensitivity toparaquat and an increase in LIPOLASE™ production. This can be explainedby the fact that manganese superoxide dismutase is a homodimer soexpression of the wild-type and truncated forms coded for by thewild-type and disrupted cassette, respectively, leads to heterodimerswhich are either non-functional or partially functional. TABLE 5 Shakeflasks 24 well results results Paraquat (Relative (Relative Strainsensitivity LU/ml) LU/ml) Southern Results HowB430 − 1.0 1.0 wild-type430162T1 − 1.0 1.1 wild-type & other 430162T2 ++ 1.3 2.5 wild-type &other 430162T3 ++ 1.4 3.0 wild-type & other 430162T4 +++ 1.2 2.1disrupted 430162T5 +++ 1.0 1.5 disrupted 430162T6 − 0.9 1.2 wild-type &other

Example 20 Characterization of Aspergillus oryzae DEBY1204.3.3 RescuedLocus pSMO1204

[0532] The Aspergillus oryzae DEBY1204.3.3 rescued locus pSMO1204containing 2.0 kb was sequenced according to the procedure described inExample 2. The nucleic acid sequence (SEQ ID NO:34) as shown in FIG. 20had no sequence homology to any published sequences.

[0533] Southern analysis and sequencing of a genomic clone was used toconfirm that no deletions had taken -place when the tagged mutant wasgenerated. A Southern blot of Aspergillus oryzae HowB430 genomic DNAdigested with various restriction endonucleases (BamHI, BglII, SalI andSphI) was prepared and analyzed as described in Example 14. Probes fromboth ends of the rescued plasmid were generated by PCR using the primersdescribed below. The primers were synthesized with an Applied BiosystemsModel 394 DNA/RNA Synthesizer according to the manufacturer'sinstructions. 970052 5′-CTATGATTGGCCGATAGG-3′ (SEQ ID NO:35) 9700535′-CCAGGCTCGCACGCTTTC-3′ (SEQ ID NO:36) 970054 5′-CTTGCAACTAACGGGGTT-3′(SEQ ID NO:37) 970055 5′-TGAGAAAGACCAAGAATG-3′ (SEQ ID NO:38)

[0534] Probe 1 was generated from one end of the rescued locus by PCRusing primer 970052 and primer 970053. Probe 2 was generated from theother end of the rescued locus by PCR using primer 970054 and primer970055. The amplification/labeling reaction (50 μl) contained thefollowing conponents: 10 ng rescued plasmid pSMO1204, 50 pmole each ofprimer 970052 and 970053 for probe 1 or 50 pmole each of primer 970054and primer 970055 for probe 2, 1×DIG labeling mix (Boehringer Mannheim,Indianapolis, Ind.), 1×Taq polymerase buffer, and 2.5 U of Taqpolymerase. The reaction was incubated in an Ericomp Thermal Cyclerprogrammed as follows: One cycle at 95° C. for 5 minutes followed by 30cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and 72° C. for1.5 minutes

[0535] Southern blots of Aspergillus oryzae HowB430 genomic DNA digestedwith various restriction enzymes were prepared and analyzed according tothe procedure described in Example 14. The blots were hybridizedindependently to probe 1 and probe 2. Identical banding patterns of thedigests with both probes would suggest that a deletion had not occurred.Conditions of the Southern analysis were as follows: Blots wereprehybridized for 1 hour and hybridized overnight at 42° C. in Easy Hyb.Blots were washed in 2×SSC, 0.1% SDS twice at room temperature for 15minutes each, then washed twice at 65° C. in 0.1×SSC, 0.1% SDS for 15minutes each. Detection continued with buffers and reagents fromBoehringer Mannheim's DIG Wash Block Buffer System. CDP star (BoehringerMannheim, Indianapolis, Ind.) was used to detect the chemiluminescentreaction. Film was exposed for approximately 1 hour.

[0536] The Southern blot results showed identical banding patterns withthe different digests suggesting a direct insertion of the taggedplasmid. A genomic clone was obtained by probing an Aspergillus oryzaeHowB430 Ziplox library obtained as described in Example 12 with theprobe from the tagged mutant Aspergillus oryzae DEBY1204. The Ziploxlibrary of Aspergillus oryzae HowB430 was screened with probe 1. Agenomic clone was isolated and sequenced (Example 2) confirming that nodeletions had occurred during the tagging event and the tagged plasmidhad inserted at an EcoRI site.

Example 21 Characterization of Aspergillus oryzae Mutant HIN603 RescuedLocus pSMO603

[0537] The Aspergillus oryzae HIN603 rescued locus pSMO603 containing1.0 kb was sequenced according to the procedure described in Example 2.The nucleic acid sequence (SEQ ID NO:39) as shown in FIG. 21 showed nohomology to any published sequences.

[0538] A Southern blot of Aspergillus oryzae HowB430 genomic DNAdigested with the restriction enymes SphI, SalI, and BamHI washybridized to probes made from both ends of the rescued plasmid. Probe 3was generated by PCR using primer 970858 and primer 970859 shown belowsynthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizeraccording to the manufacturer's instructions. Probe 4 was generated byPCR using primer 970860 and primer 970861. The template for the 50 μlPCR labeling reaction was 10 ng of the rescued plasmid pSMOH603. PCRcycles and conditions were as described in Example 20. Southernconditions were as described in Example 20. 970858:5′-TGTAGTCTGACTAGCATG-3′ (SEQ ID NO:40) 970859: 5′-GGATCTTCACCTAGATCC-3′(SEQ ID NO:41) 970860: 5′-CATAGTGTCGACCAAGC-3′ (SEQ ID NO:42) 970861:5′-CAATCGAGCTTGCCTATG-3′ (SEQ ID NO:43)

[0539] Different banding patterns on the Southern suggested that adeletion had taken place where the tagging occurred. When 500 ng ofAspergillus oryzae HowB430 genomic DNA was used as a template in a PCRreaction with primers 090858 and 090860, a 3 kb product was amplifiedsuggesting a 2.5 kb deletion had occurred. Southern analysis of genomicDNA prepared from the tagged Aspergillus oryzae HIN603 strain, digestedwith HindIII, and probed with the NheI fragment from Aspergillus oryzaepyrG suggested that the tagged mutant had not been generated by a REMIevent.

Example 22 Construction of Aspergillus oryzae HowB432

[0540]Aspergillus oryzae HowB432 was generated by transformation ofAspergillus oryzae JaL250 with a linear fragment containing the NA2-tpipromoter, a cellulase gene from Humicola lanuginosa (CAREZYME™ gene,Novo Nordisk A/S, Bagsvaerd, Denmark), and the AMG terminator obtainedfrom plasmid pGAG3 (FIG. 22).

[0541]Aspergillus oryzae JaL250 was constructed from Aspergillus oryzaeJaL142 (Christensen et al., 1988, Bio/Technology 6: 1419-1422) bydeleting the neutral protease I gene (npI). The npI deletion plasmid wasconstructed by exchanging a 1.1 kb BalI fragment coding for the centralpart of the npI gene in plasmid pJaL389 (FIG. 23), which contained a 5.5kb SacI genomic fragment encoding the npI gene, with a 3.5 kb HindIIIfragment from pJaL335 (FIG. 24) containing the pyrG gene flanked byrepeats, thereby creating plasmid pJaL399 (FIG. 25). Aspergillus oryzaeJaL142 was transformed with the 7.9 kb SacI fragment. Transformants wereselected by relief of the uridine requirement on Minimal medium plates.The transformants were analyzed by Southern analysis as described inExample 14 and by IEF protease profile analysis according to standardmethods.

[0542] Two out of 35 transformants possessed an altered Southern profilecompared to the parent strain and displayed no neutral protease Iactivity by IEF. Furthermore, Southern analysis showed that one of thetwo transformants had a clean deletion of the npI gene and wasdesignated Aspergillus oryzae JaL228.

[0543] Totally, 2.3×10⁷ conidiospores of Aspergillus oryzae JaL228 werespread on Minimal medium plates supplemented with 0.1% 5-fluoro-oroticacid (FOA) and 10 mM uridine. Eight FOA resistant colonies wereobtained. A Southern blot of BamHI digested genomic DNA from the eightcolonies probed with a 401 bp pyrG repeated region demonstrated that thepyrG gene had been excised by recombination at the repeated regions.Aspergillus oryzae JaL228 showed two bands of the expected size of 2.7and 3.1 kb originating from the two copies of the repeated region. Ifthe pyrG gene had been lost by recombination between the repeatedregions, the 3.1 kb band would have disappeared and only the 2.7 kbwould have remained. All 8 FOA resistant colonies showed this pattern ofbands. Sequencing of a PCR fragment covering the junctions between thenpI gene and the copy of the 401 bp repeat remaining in the 8 coloniesconfirmed that the pyrG gene was excised by recombination between therepeats. One of the colonies was designated Aspergillus oryzae JaL250.

[0544] pGAG3 was constructed by isolating from pDM176 (FIG. 26) aSwaI/PacI fragment containing the Humicola lanuginosa cellulase gene andligating the fragment into SwaI/PacI digested pBANe6. The SwaI/PacIfragment from pDM176 and SwaI/PacI digested pBANe6 were separated on a1% agarose gel, and isolated using a QIAquick Gel Extraction Kit (QiagenInc., Chatsworth, Calif.) according to the manufacturer's instructionsprior to ligation. The ligation was used to transform E. coli DH5αcells, and the transformants were then screened by extracting plasmidDNA from the transformants using a QIAwell-8 Plasmid Kit according tothe manufacturer's instructions, restriction digesting the plasmid DNAto confirm the presence of the correct size fragment, and sequencing theDNA according to the method described in Example 2.

[0545] pGAG3 was then digested with PmeI and the linear expressioncassette was isolated by preparative agarose electrophoresis using TAEbuffer. The linear cassette was then used to transform Aspergillusoryzae JaL250.

[0546] Transformation of Aspergillus oryzae JaL250 for amdS selectionwas conducted with protoplasts at a concentration of 2×10⁷ protoplastsper ml prepared as described in Example 2. Ten μg of the linear fragmentdescribed above were added to 100 μl of protoplasts. A volume of 250 μlof PEG (60% PEG 4000-10 mM CaCl₂-10 mM Tris-HCl pH 8.0) was then added,and the mixture was placed at 37° C. for 30 minutes. Three ml of STCmedium was added and the mixture was plated on Cove plates supplementedwith 10 mM uridine for amdS selection. The plates were incubated 7-10days at 34° C. Transformants were then transferred to plates of the samemedium and incubated 3-5 days at 37° C. The transformants were purifiedby streaking spores and picking isolated colonies using the plates ofthe same medium without sucrose.

Example 23 Aspergillus oryzae Transformation with HpaI LinearizedpDSY112 and Cellulase Expression Screening

[0547]Aspergillus oryzae HowB432 was transformed with HpaI digestedpDSY112 and the transformants were recovered using the methods describedin Example 5. Totally, 104 recovered transformants were grown in 24 wellmicrotiter plates in ¼ strength MY25. Samples were taken at 5 and 7 daysand assayed for cellulase activity as described below.

[0548] Cellulase activity was measured according to the followingprotocol which is derived from Novo Nordisk method AF 302.1/1-GB whichis available from Novo Nordisk A/S, Bagsvaerd, Denmark upon request. Asubstrate solution containing 2% azo-carboxymethylcellulose was preparedby dissolving the material in 100 mM MOPS pH 7.0 buffer at 80° C. for 10minutes. CAREZYME™ (Novo Nordisk A/S, Bagsvaerd, Denmark) was used as astandard. Stock solutions of 2.5 to 25 ECU per ml were prepared toconstruct a standard curve by diluting accordingly CAREZYME™ in 100 mMMOPS pH 7.0 buffer. Five μl aliquots of the standards and samples(diluted for shakeflasks and fermentations) were pipetted intoindividual wells of a 96 well plate. A volume of 65 μl of the 2%azo-carboxymethylcellulose solution was pipetted into each of the wellsand mixed. The reactions were incubated at 45° C. for 30 minutes andthen stopped by the addition of 215 μl of stop reagent followed bymixing. The stop reagent was prepared by first suspending 0.2 g of ZnCl₂in 20 ml of 250 mM MOPS pH 7.0 and adding the suspension to 80 ml ofacidified ethanol containing 1.1 ml of concentrated HCl per liter ofethanol. The plate containing the stopped reaction was then centrifugedat 3000 rpm for 10 minutes. A 100 μl aliquot of each supernatant waspipetted into a 96 well plate and the absorbance measured at 600 nm.Using linear regression, the slope, intercept, and correlationcoefficient were determined for the standards and samples.

[0549] The top ten cellulase producing transformants from the 24 wellcultures were spore purified, and regrown in 24 well cultures as aboveand assayed for cellulase activity. The purified strains were also grownin MY25 in 125 ml shake flasks in MY25 pH 6.5 at 34° C. and samples weretaken at 3 and 5 days for cellulase assays. Aspergillus oryzae HowB432pDSY112 84-1-1 and Aspergillus oryzae HowB432 pDSY112 94-1-1 were alsogrown in fermentors (2 liters) as described in Example 8. Cellulaseactivities were measured as described above.

[0550] The results from the 24 well and shake flasks cultures arepresented in Table 6 where the cellulase yield of Aspergillus oryzaeHowB432 was normalized to 1.0. TABLE 6 24 well Shake flasks Fermentationresults Strain (ECU/ml) (ECU/ml) (ECU/ml) HowB432 1.0 1.0 1.0C112T50.1.1 2.0 0.9 NA C112T84.1.1 2.1 2.0 1.3 C112T86.1.1 2.3 2.4 NAC112T94.1.1 2.5 1.3 1.4 C112T95.1.1 2.1 1.3 NA C112T100.1.1 2.3 1.6 NAC112T101.1.1 1.9 1.6 NA C112T102.1.1 2.0 2.1 NA C112T103.1.1 1.5 2.0 NAC112T104.1.1 2.2 2.3 NA

[0551] A Southern blot of Aspergillus oryzae HowB432, Aspergillus oryzaeDEBY599.3 and the pDSY112 transformants genomic DNAs digested with BglIIwas prepared and analyzed as described in Example 14 performed todetermine whether pDSY112 had integrated at the homologous locus in thegenome using the Aspergillus oryzae DEBY599.3 rescued flanking DNA as aprobe.

[0552] A BglII band of 2.7 kb from Aspergillus oryzae HowB432 hybridizedwith the probe, while an ˜8 kb BglII band from Aspergillus oryzaeDEBY599.3 hybridized to the probe. In all of the transformants awild-type BglII band of 2.7 kb and a second band corresponding to thetransforming DNA hybridized to the probe. Therefore, none of theretransformants had exact gene replacements.

Example 24 Aspergillus oryzae Transformation with NdeI LinearizedpDSY138 and Cellulase Expression Screening

[0553]Aspergillus oryzae HowB432 was transformed with NdeI digestedpDSY138 using the method described in Example 5. Totally, 240transformants were recovered which were grown in 24 well microtiterplates in ¼ strength MY25 as described in Example 8 except samples weretaken at days 3 and 5 and assayed for cellulase activity as described inExample 23. The top 20 cellulase producing transformants were sporepurified and retested in 24 well microtiter cultures. The top 8cellulase producing once purified transformants were spore purified asecond time and tested in shake flasks in full-strength MY25 asdescribed in Example 8. The top 2 producers were also grown in a 2 literfermentor as described in Example 8. Cellulase activity was measured asdescribed in Example 23.

[0554] The results obtained are shown in Table 7 below where thecellulase yield of Aspergillus oryzae HowB432 is normalized to 1.0.TABLE 7 Strain ECU/ml Southern Results HowB432 1.0 wild-type C138T21.1.11.15 disrupted allele C138T205.1.1 1.5 wild-type and disrupted alleles

[0555] A Southern blot of Aspergillus oryzae DEBY932, Aspergillus oryzaeHowB432, and pDSY138 genomic DNA preparations digested with NdeI wasprepared and analyzed as described in Example 14 to determine if thepDSY138 DNA had integrated at the homologous locus producing genereplacements in the transformants using the Aspergillus oryzae DEBY932rescued flanking DNA as a probe.

[0556] The results of the Southern blot demonstrated that an NdeI bandof approximately 5 kb from Aspergillus oryzae HowB432 hybridized to therescued locus while a band of approximately 10 kb from Aspergillusoryzae DEBY932 hybridized to the probe. In Table 7, the column labeledSouthern results indicated whether the transformants had a wild-typeNdeI fragment of the size observed in the parent strain Aspergillusoryzae HowB432 or whether the transformants had a band corresponding tothe disrupted size observed in Aspergillus oryzae DEBY932.

Example 25 Aspergillus oryzae Transformation with AseI/PvuI palBDisruption Cassette from pMT1936 and Cellulase Screening

[0557]Aspergillus oryzae HowB432 was transformed using the sametransformation procedure described in Example 5 with a 5.5 kb AseI/PvuIfragment containing the palB disruption cassette. The linear fragmentfor transformation was isolated by digestion of pMT1936 with AseI andPvuI and separation of the fragment on a 1% agarose gel using a QIAquickGel Extraction Kit according to the manufacturer's instructions. Thetransformants obtained were then evaluated for growth on Minimal mediumplates at pH 6.5 or pH 8.0.

[0558] The results showed that 10 of the 312 transformants tested wereunable to grow at pH 8.0 indicative of the palB minus phenotype. The 10palB minus transformants and 10 of the transformants that were able togrow at pH 8.0 were spore purified and tested in 24 well and shake flaskcultures for cellulase production according to the procedures describedin Example 23. The results tabulated in Table 8 below demonstrated thatpalB minus strains were better cellulase producers than the palB plusstrains.

[0559] Southern blots of the genomic DNA from an Aspergillus oryzae palBminus mutant, an Aspergillus oryzae palB plus strain, and Aspergillusoryzae HowB432 were performed as described in Example 14 to determine ifthe AseI/PvuI transforming DNA fragment had integrated as a cleanreplacement into the palB locus.

[0560] The results of the Southern blot (Table 8) demonstrated that someof the palB minus strains had clean disruptions while others did not.TABLE 8 Shake flasks Strain PalB phenotype (ECU/ml) Southern patternHowB432 plus 1.0 wild-type CpalB5-1 plus 1.0 wild-type and otherCpalB6-1 plus 1.2 wild-type and other CpalB7-1 plus 1.1 wild-type andother CpalB24-1 plus 1.7 wild-type and other CpalB28-1 minus 1.6 otherCpalB34-1 minus 1.6 disruption CpalB45-1 minus 1.4 disruption CpalB47-1plus 1.1 wild-type and other CpalB72-1 plus 1.3 wild-type and otherCpalB76- 1 minus 1.6 disruption CpalB89-1 minus 1.6 disruptionCpalB153-1 minus 1.1 disruption CpalB161-1 plus 1.2 wild-type and otherCpalB163-1 minus 1.9 other CpalB185-1 minus 1.8 other CpalB190-1 minus1.4 disruption

Example 26 Aspergillus oryzae Transformation with AsnI/PvuI ManganeseSuperoxide Dismutase Disruption Cassette and Cellulase Screening

[0561]Aspergillus oryzae HowB432 was transformed with a 5.8 kb AsnI/PvuIfragment containing the manganese superoxide dismutase disruptioncassette according to the same procedure described in Example 5.

[0562] Twenty transformants were obtained and tested for sensitivity toparaquat as described in Example 17. Seven of the 20 transformants wereparaquat sensitive indicative of the manganese superoxide dismutaseminus phenotype although they are sensitive to different levels ofparaquat as indicated in Table 9 below. Those indicated as +++ forparaquat sensitivity are sensitive to as low as 2 mM paraquat, whilethose labeled − and ++ are not sensitive to paraquat and sensitive tointermediate levels of paraquat, respectively. All of the transformantswere spore purified and tested in 24 well cultures for cellulaseproduction as described in Example 23. The strains were also tested in125 ml shake flasks cultures as described in Example 23. The results areshown in Table 9 below. The strains that are paraquat sensitive produceon average more CAREZYME™ than those strains that are not paraquatsensitive.

[0563] Southern blots of the transformants and Aspergillus oryzaeHowB432 were prepared and analyzed as described in Example 14 todetermine if the AsnI/PvuI manganese superoxide dismutase disruptioncassette had integrated to give a clean replacement into the manganesesuperoxide dismutase disruption cassette locus.

[0564] The results of the Southern blot shown in Table 9 below indicatethat the strains sensitive to 2 mM paraquat have only the disruptedlocus of manganese superoxide dismutase, while those sensitive tointermediate levels of paraquat have both the wild-type locus and thedisrupted cassette. The intermediate sensitivity to paraquat may beexplained by the fact that manganese superoxide dismutase is ahomodimer, and those cells expressing the wild-type and truncatedmanganese superoxide dismutase coded for by the disruption cassettewould be producing heterodimers that are probably not functional. TABLE9 Shake flasks Strain Paraquat sensitivity (Relative LU/ml) SouthernResults HowB432 − 1.00 wild-type 432162T3 +++ 1.26 disrupted 432162T7 ++1.24 wild-type & other 432162T8 − 1.21 wild-type & other 432162T9 − 0.61wild-type & other 432162T10 ++ 1.14 wild-type & other 432162T11 ++ 1.04wild-type & other 432162T12 ++ 1.12 wild-type & other 432162T15 − 0.44wild-type & other 432162T16 ++ 1.10 wild-type & other 432162T17 +++ 1.53— 432162T18 − 1.12 — 432162T19 +++ 1.44 —

Example 27 Construction of Glucose Transporter Gene OverexpressionPlasmids pHB218 and pDSY153 and Stop Control Plasmids pDSY152 andpDSY155

[0565] Plasmids to overexpress the glucose transporter rescued locusfrom Aspergillus oryzae DEBY599.3 were constructed to determine ifoverexpression of the glucose transporter would lead to an increase inthe yields of Humicola lanuginosa lipase and cellulase. The glucosetransporter open reading frame was PCR amplified to place SwaI and PacIsites at the 5′ and 3′ end of the ORF, respectively. The followingprimers synthesized with an Applied Biosystems Model 394 DNA/RNASynthesizer according to the manufacturer's instructions were used incombination with 0.2 μg of pDSY112 in the amplification: 961176:5′-ATTTAAATGGTCCTCGGTGGATCAAGC-3′ (SEQ ID NO:44) 961177:5′-TTAATTAATTAGTCCTGTCTGCGCTGGT-3′ (SEQ ID NO:45)

[0566] The conditions and parameters used for the amplification aredescribed in Example 2. Ten μl of the PCR reaction was electrophoresedon an agarose gel, and a 1.5 kb product was obtained as expected. ThePCR product was cloned using a pPCR-Script™ Kit (Stratagene, La Jolla,Calif.) according to the manufacturer's protocols. The ligation reactionwas used to transform E. coli DH5α cells, and plasmid DNA was isolatedfrom several of the tramsformants using the QIAWell-8 Plasmid Kit. Theplasmids were digested with NotI and EcoRi to determine which clones hadthe 1.5 kb insert. Six of the 11 clones analyzed had the correct sizeinsert as determined by electrophoreses on an agarose gel. One of theclones, pDSY119, was digested with PacI and SwaI, and the digest was runon an agarose gel. The 1.5 kb SwaI/PacI band was excised from the gel,and DNA was purified from the gel slice using the QIAQuick GelExtraction Kit. The 1.5 kb fragment was ligated with SwaI/PacI cutpBANe13 (FIG. 3) using standard conditions (Sambrook et al., 1989,supra). The ligation was used to transform E. coli DH5α cells, andplasmid DNA was isolated from several of the transformants. The plasmidswere digested with SwaI/PacI to determine which clones had the expected1.5 kb insert. The final plasmid was designated pHB218 (FIG. 27).

[0567] As a control for the overexpression experiments, a derivative ofpHB218 in which a stop codon was inserted at amino acid 9 in the glucosetransporter open reading frame was made using site-directed mutagenesis.A MORPH™ Site-Specific Plasmid DNA Mutagenesis kit from 5 Prime→3 Primewas used for the mutagenesis, and protocols provided with the kit werefollowed. The reaction contained pHB218 as template, and the mutagenicprimer used was:

970545: 5′-CGGTGGATCAAGCGGTTAATTAATCACTCCGTACCTGAT-3′  (SEQ ID NO:46)

[0568] Several E. coli colonies were obtained after following theprotocols, and plasmid DNA was isolated from the colonies using theQIA-Well8 plasmid kit. The plasmids were digested with PacI since themutagenic primer introduced a PacI site which served as a marker for themutagenesis. Two of the plasmids with the extra PacI site indicative ofa successful mutagenesis were sequenced as described in Example 2 toconfirm the presence of the stop codon at amino acid 9 in the ORF. ThepHB218 derivative with the stop codon at amino acid 9 was designatedpDSY152.

[0569] Versions of pHB218 and pDSY152 in which the selectable marker wasthe bar gene were constructed for transformation of strains which arepyrG plus. The SwaI/PacI inserts from pHB218 and pDSY152 were isolatedby restriction digestion, electrophoresed on an agarose gel, andpurified using QIAQuick Gel Extraction Kit. The inserts were ligatedinto pSE39 (FIG. 28) and digested with SwaI/PacI. The ligation reactionwas used to transform E. coli DH5α, and plasmid DNA was isolated fromthe colonies as described above. The plasmids were digested withSwaI/PacI to determine which clones contained the expected 1.5 kbinsert. The plasmids were sequenced as described in Example 2 to confirmthe presence or absence of the stop codon at amino acid 9 in pDSY155 andpDSY153 (FIG. 29), respectively. The only difference between pDSY155 andpDSY153 was the stop codon at amino acid 9 of the glucose transporterORF in pDSY155.

Example 28 Transformation of Aspergillus oryzae HowB430 and Aspergillusoryzae HowB432 with pHB218 and pDSY152 and lipase and cellulaseScreening, Respectively.

[0570]Aspergillus oryzae HowB430 was transformed with pHB218 or pDSY152,and the transformants were recovered using the methods described inExample 5. One hundred and twenty transformants each with pHB218 andpDSY152 were recovered, grown in 24-well microtiter plates in {fraction(1/100)} strength MY25 and assayed for lipase activity after 3 and 5days as described in Example 8. The assay results showed that there wasa slight shift towards higher lipase production in the pHB218transformants versus the pDSY152 transformants supporting the idea thatoverexpression of the glucose transporter has a positive effect onlipase expression.

[0571]Aspergillus oryzae HowB432 was transformed with pHB218 andpDSY152, and the transformants were recovered using the methodsdescribed in Example 5. One hundred transformants each with pHB218 andpDSY152 were recovered, grown in 24-well microtiter plates in ¼ strengthMY25 and assayed for cellulase activity after 3 and 5 days as describedin Example 23. The assay results showed that there was a shift towardshigher cellulase production in the pHB218 transformants versus thepDSY152 transformants indicating that overexpression of the glucosetransporter had a positive effect on cellulase expression.

Example 29 Transformation of Aspergillus oryzae DEBY10.3 with pDSY153and pDSY155 and lipase Screening.

[0572]Aspergillus oryzae DEBY10.3 was transformed with pDSY153 andpDSY155, and the transformants were recovered using the methodsdescribed in Example 5. Two hundred sixteen and 144 transformants withpDSY153 and pDSY155, respectively, were recovered, grown in 24-wellmicrotiter plates in {fraction (1/100)} strength MY25, and assayed forlipase activity on days 4 and 6 as described in Example 8. There wasshift towards higher lipase production in the pDSY153 transformants whencompared to the pDSY155 transformants indicating that overexpression ofthe glucose transporter led to an increase in lipase production and alsosuggesting that the palB minus effect and the glucose transporteroverexpression were additive.

Example 30 Identification of Tagged Event in Aspergillus oryzae HowL795

[0573] Genomic DNA was prepared from Aspergillus oryzae HowL795according to Example 9. One μg of DNA was digested with either SnaB1 orNsiI. Both enzymes cleave within the pyrG gene contained on the taggingconstruct. The DNA was then diluted to 4 ng/μl and recircularized withT4 Ligase at 22° C. for 18 hours. Inverse PCR was then performed usingapproximately 500 ng of recircularized DNA using the primers shown belowwhich were synthesized with an Applied Biosystems Model 394 DNA/RNASynthesizer according to the manufacturer's instructions. Both werelocated downstream of the NsiI and SnaBI sites. Primer x:5′-GCACTCGAATGACTACT-3′ (SEQ ID NO:47) Primer y:5′-CGCATCATACTTGCGACA-3′ (SEQ ID NO:48)

[0574] The inverse PCR amplification reaction contained the followingcomponents: 500 ng of recircularized DNA, 150 pmoles of primer x, 150pmoles of primer y, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taqpolymerase buffer, and 2.5 U of Taq polymerase. The reaction wasincubated in an Ericomp Thermal Cycler programmed as follows: One cycleat 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1minute, 55° C. for 1 minute and 72° C. for 2 minutes. The PCR productwas isolated by electrophoresis on a 1% agarose gel.

[0575] PCR of the religated SnaBI DNA amplified a 4 kb fragment whereasPCR of the religated NsiI DNA amplified a 2 kb fragment. PCR confirmedthat the smaller NsiI fragment was contained within the larger SnaBIfragment.

[0576] DNA sequence analysis was performed according to the proceduredescribed in Example 2 using primer A. The analysis identified that theinsertion of pSO122 had occurred in the 3′ non-translated region of theamdS gene contained within plasmid pBANe8.

Example 31 Construction of Aspergillus oryzae MStr107

[0577]Aspergillus oryzae MStr107 was constructed to contain extra copiesof one of the native alpha-amylase (TAKA) genes (FUNGAMYL™ gene, NovoNordisk A/S, Bagsvaerd, Denmark), by transforming Aspergillus oryzaeHowB101 with a DNA fragment from pMStr15. pMStr15 was constructed frompCaHj505 and pTAKA17 as described below. Standard methods were employed(Sambrook et al., 1989, supra) except where noted.

[0578] pCaHj505 (FIG. 30) was constructed to contain the Aspergillusoryzae NA-14 alpha-amylase (TAKA) promoter, the Aspergillus nigerglucoamylase (AMG) terminator, and the Aspergillus nidulans amdS genefrom the following fragments:

[0579] a) The vector pToC65 (WO 91/17243) digested with EcoRI and XbaI.

[0580] b) A 2.7 kb XbaI fragment from Aspergillus nidulans carrying theamdS gene (Corrick et al., 1987, Gene 53: 63-71). The amdS gene was usedas a selective marker in fungal transformations. The amdS gene wasmodified so that the BamHI site normally present in the gene wasdestroyed. This was done by introducing a silent point mutation usingthe primer: AGAAATCGGGTATCCTTTCAG (SEQ ID NO:49).

[0581] c) A 0.6 kb EcoRI-BamHI fragment carrying the Aspergillus oryzaeNA-14 alpha-amylase promoter.

[0582] d) A 675 bp xbaI fragment carrying the Aspergillus nigerglucoamylase transcription terminator. The fragment was isolated fromthe plasmid pICAMG/Term (EP 238 023).

[0583] The BamHI site of fragment c was connected to the XbaI site infront of the transcription terminator on fragment d via the pIC19Rlinker (BamHI to XbaI) (Boehringer Mannheim, Indianapolis, Ind.).

[0584] pMStr15 (FIG. 31) was constructed to contain the Aspergillusoryzae NA-14 alpha-amylase promoter, gene and terminator and theAspergillus nidulans amdS gene. The alpha-amylase gene with promoter andterminator was excised from pTAKA17 (European patent 0238 023) as a 2.9kb EcoRI-HindIII fragment and cloned adjacent to the amdS gene in thevector pCaHj505 by replacing the EcoRI-XbaI promoter/terminator fragmentin pCaHj505. To facilitate cloning, the recessed 3′ termini generated byHindIII and XbaI digestion were filled in.

[0585] A single linear DNA fragment containing both the alpha-amylasegene and the amdS gene was obtained by digesting pMStr15 with NotI,resolving the vector and insert sequences using agarose gelelectrophoresis, excising the appropriate DNA band from the gel, andpurifying the DNA from the agarose using GenElute™ Agarose Spin Columnsaccording to the manufacturer's directions (Supelco, Bellefonte, Pa.).This 5.6 kb NotI fragment was used to transform Aspergillus oryzaeHowB101 to construct Aspergillus oryzae MStr107, using thetransformation protocol and selective medium described in Example 2.Transformants were propagated from single colonies twice in successionon COVE medium with 0.1% Triton X100 before performing additionalscreens.

[0586]Aspergillus oryzae MStr107 was selected from among thetransformants based on its ability to produce more alpha-amylasethanAspergillus oryzae HowB101. The ability of the transformants toproduce alpha-amylase was determined by culturing them in 10 ml of YPMmedium for 4 days at 30° C. with shaking and resolving 5 μl of theculture medium by SDS-PAGE according to standard methods. The strainproducing the most alpha-amylase under these conditions was selected asAspergillus oryzae MStr107, and was compared in a 3 liter fermentationculture to Aspergillus oryzae HowB101. The medium was composed ofmaltose syrup, yeast extract, KH₂PO₄, K₂SO₄, (NH₄)₂SO₄, citric acid,MgSO₄, trace metals and uridine. Under these conditions, Aspergillusoryzae MStr107 produced 360% of Aspergillus oryzae HowB101.

Example 32 Aspergillus oryzae Mstr107 Transformation with LinearizedpDSY82

[0587] Protoplasts of Aspergillus oryzae Mstr107 were prepared asdescribed in Example 2. A 5-15 μl aliquot of pDSY82 (6 μg) linearizedwith 1.25 U of XbaI was added to 0.1 ml of the protoplasts at aconcentration of 2×10⁷ protoplasts per ml in a 14 ml Falconpolypropylene tube followed by 250 μl of 60% PEG 4000-10 mM CaCl₂-10 mnMTris-HCl pH 7, gently mixed, and incubated at 37° C. for 30 minutes.Three ml of SPTC were then added and the suspension was gently mixed.The suspension was mixed with 12 ml of molten overlay agar (1×COVEsalts, 1% NZ amine, 0.8 M sucrose, 0.6% Noble agar) or 3 ml of STCmedium and the suspension was poured onto a Minimal medium plate. Theplates were incubated at 37° C. for 3-5 days.

[0588] The transformation frequency of Aspergillus oryzae MStr107 withpDSY82 and XbaI was approximately 200 transformants/μg of DNA. A libraryof approximately 30,000 transformants was obtained. Spores from 70 poolswith approximately 400 transformants in each pool were collected andstored in a 20% glycerol, 0.1% Tween 80 at −80° C. The pools andtransformants from these libraries were designated with the letter “x”.

Example 33 Characterization of Integration Events in “REMI” Aspergillusoryzae MStr107 Transformants

[0589] Transformants of Aspergillus oryzae MStr107 with pDSY82 and XbaI(library “x”) were analyzed as described in Example 6. Genomic DNA wasisolated from 40 transformants, 20 from one pool (x15) and 20 from 20various pools. DNA samples were cut with HindIII, resolved, blotted andprobed with radiolabeled pDSY82. Thirty-three of 40 displayed apparentlynovel band patterns, suggesting that plasmid integrations weredistributed to different sites in the genome. For 19 of the 40transformants the band patterns suggested that only one copy of pDSY82integrated in the genome, while more than one copy was observed in theremaining 21 transformants. DNA from the 20 transformants taken fromvarious pools was also cut with XbaI, resolved, blotted and probed withradiolabeled pDSY82 as described in Example 6. A single, plasmid-sizedband was observed indicating REMI had occurred at an XbaI site in 9 ofthe transformants.

Example 34 FUNGAMYL™ Expression Screening

[0590] The Aspergillus oryzae MStr107 tagged mutant library “x” poolsdescribed in Example 32 were assayed for FUNGAMYL™ expression.

[0591] For 96-well plate screens, MTBCDYU medium was used. For 24-wellplate methods, 4×MTBCDYU medium was used.

[0592] Primary 96-well plate screens involved the dilution of sporesfrom distinct pools into MTBCDYU so that one spore on average wasinoculated per well when 100 μl of medium was dispensed into the wells.After inoculation, the 96-well plates were grown for 3-4 days at 34° C.under static conditions. Cultures were then assayed for FUNGAMYL™activity as described below. Mutants of interest were isolated twice onYPG or Cove plates, and single colonies transferred to Cove agar slants.Spores from Cove slants were inoculated into 24-well plates containing4×MTBCDYU with approximately 103 spores per well and grown under staticconditions for 4 days at 34° C. Cultures were then assayed for FUNGAMYL™activity as described below.

[0593] The FUNGAMYL™ assay substrate(4-nitrophenyl-alpha-D-maltoheptasid-4,6-O-ethyliden, EPS) was preparedas a ½ strength solution relative to the instructions given by themanufacturer (Boehringer Mannheim, Indianapolis, Ind.). The substratewas prepared in HEPES pH 7.0 buffer. A FUNGAMYL™ standard (FUNGAMYL™,Novo Nordisk A/S, Bagsvaerd, Denmark) was prepared to contain 2 FAU/mlin HEPES pH 7.0 buffer. The standard was stored at −20° C. until use.FUNGAMYL™ stock was diluted appropriately to obtain a standard seriesranging from 0.02 to 0.2 FAU/ml just before use. Broth samples werediluted in HEPES buffer and 25 μl aliquots were dispensed to wells in96-well plates followed by 180 μl of diluted substrate. Using a platereader, the absorbance at 405 nm was recorded as the difference of tworeadings taken at approximately 1 minute intervals. FUNGAMYL™ units/ml(FAU/ml) were calculated relative to the FUNGAMYL™ standard solutions.

[0594] The results of the 96-well screen followed by the 24-well screenidentified for further evaluation 51 transformants from the pDSY82 andXBaI transformations. These identified transformants produced higherlevels of FUNGAMYL™ than the control strain Aspergillus oryzae MStr107.

Example 35 Shake Flask and Fermentation Evaluation

[0595] The highest FUNGAMYL™-producing DNA-tagged mutants described inExample 34 were evaluated in shake flasks and fermentors.

[0596] Shake flask evaluations were performed by inoculating half thespore content of a COVE slant suspended in a suitable volume of sterilewater containing 0.02% TWEEN-80 into 100 ml of G1-gly medium at pH 7.0in a 500 ml shake flask. The G1-gly shake flasks were incubated at 34°C. for 1 day at 270 rpm. Next, 5 ml of the G1-gly cultures wereinoculated into 100 ml of ⅕MDU2BP at pH 6.5 in 500 ml shake flasks.Samples were taken at day 3, and FUNGAMYL™ activity was measured asdescribed in Example 34.

[0597] The DNA-tagged mutant X70-25 and 257D11 were grown in a 3 literlab fermentor containing a medium composed of Nutriose, yeast extract,MgSO₄, KH₂PO₄, citric acid, K₂SO₄, (NH₄)₂SO₄ and trace metals solution.The fermentation was performed at a temperature of 34° C., a pH of 7,and the agitation was maintained between 1000-1200 rpm for 5 days.FUNGAMYL™ activity was measured by partial degradation of EPS to G₂ andG₃ derivatives (G=glucose). The G₂ and G₃ derivatives were then degradedto glucose and yellowish colored p-nitrophenolate anion by the additionof a surplus of alpha-glucosidase. The analytical output was determinedas the change in absorbance at 405 nm per unit time (3 minutes) at 37°C. and pH 7.1 after a preincubation for 2.5 minutes. FUNGAMYL™ was usedas standard.

[0598] The results obtained are shown in Table 10 below where theFUNGAMYL™ yield of Aspergillus oryzae MStr107 as a control is normalizedto 1.0. TABLE 10 FUNGAMYL ™ Expression by DNA Tagged Mutants 24-wellShake Flask in 96-well Plate Fermentation Strain # Plates ResultsResults Description Pool Screened (FAU/ml) (FAU/ml) (FAU/ml) HowB101 NANA 0.4 0.4 0.3 MStr107 NA NA 1.0 1.0 1.0 X70-25 x70 580 1.4 1.3 1.2257D11 x6 480 1.5 1.4 1.0 X70-42 x70 580 1.3 1.3 ND 263A3 x6 480 1.4 1.6ND X69-246 x69 350 1.6 1.4 ND X59-122 x59 580 1.3 1.3 ND X49-233 x49 4601.4 1.4 ND

[0599] As shown in Table 10, the mutants produced approximately 30-60%more FUNGAMYL™ than the control strain Aspergillus oryzae MStr107 whengrown in 24-well plates and when grown in shake flasks. The mutant,Aspergillus oryzae X70-25 produced approximately 20% more FUNGAMYL™ thanthe control strain Aspergillus oryzae MStr107 when grown in fermentors.

Example 36 Screening for Morphological Mutants

[0600] The 5 “e” pools and 5 “b” pools described in Example 5 werescreened for altered morphology by plating on CM-1 agar and incubatingat 34° C. for 4 days.

[0601] Twenty-four colonies having altered plate morphology and coveringthe morphological variation within the pool were transferred to freshCM-1 plates and incubated 5 days at 34° C. for single colony isolation.Each morphology (in most cases 1) on a plate, was transferred from asingle colony to the center of a CM-1 plate and a PDA plate, andincubated 6-8 days at 34° C. before the morphology was evaluated, i.e.,the diameter and the appearance. A total of 218 morphological mutantswas transferred to COVE plates and incubated at 34° C. for 1-2 week togenerate spores.

Example 37 Evaluation of Morphological Mutants

[0602] The morphological mutants isolated in Example 36 were evaluatedin 24-well plates for lipase production according to the proceduredescribed in Example 7. The highest yielding mutants were compared withrespect to plate morphology on CM-1 agar, and 23 mutants covering theobserved morphological variation were further tested in shake flaskscontaining CD medium to evaluate lipase production.

[0603] Approximately 0.25 ml of spore suspension from a CM-1 plate wasinoculated into 25 ml of G1-gly medium in a 125 ml PP flask andincubated at 34° C. for 24 hours. Then 0.5 ml of the 24 hour seed flaskwas transferred to 50 ml of CD medium supplemented with 1.0 μl ofFUNGAMYL™ 800L (Novo Nordisk A/S, Bagvaerd, Denmark) in a 125 ml PPflask, and incubated at 34° C., 200 rpm. The culture was sampled after 2and 3 days and assayed for lipase activity as described in Example 7.

[0604] The isolated mutants were also tested in the following manner inoxygen limited media. Aspergillus oryzae HowL536.3 was run as a controlsince the strain possessed the wild type morphology and did not requireuridine for growth. Approximately 250 μl of spore suspension wasinoculated into a 125 ml shake flask containing 25 ml OL-1 medium andincubated at 34° C., 200 rpm until residual glucose was<<1 g/l measuredusing DIASTIX™ (Bayer, Elkhart, Ind.). Then 75 ml OL-6 medium was addedto each flask and further incubated at 34° C., 200 rpm for approximately25 hours until residual glucose in the Aspergillus oryzae HowL536.3flask was approximately 5 g/l. At that time, all the flasks were assayedfor residual glucose, and the flasks with significantly lower glucose(0-2 g/l) were considered positive. The majority of the flasks averagedaround 5-10 g/l.

[0605] Twenty mutants converting the glucose faster than average wereconsidered likely to be easier to aerate and were further tested inshake flasks containing CD medium as described above to evaluate lipaseexpression.

[0606] Based on these tests, 14 mutants were identified and furtherevaluated in lab fermentors according to the procedure described inExample 8. The morphological mutants listed below in Table 11 wereidentified. TABLE 11 Morphological mutants Strain Construction PoolDescription P2-7.1 pDSY82 + BamHI b2 colonial, easy to aerate, 50% yieldincrease in fermentors P3-2.1 pDSY82 + BamHI b3 flat, yield 40% yieldincrease in fermentors P4-8.1 pDSY82 + BamHI b5 easy to aerate, 30%yield increase in fermentors P5-7.1 pDSY82 + BamHI b6 easy to aerate,60% yield increase in fermentors P7-14.1 pDSY81 + EcoRI e2 colonial,easy to aerate, 50% yield increase in fermentors P8-10.1 pDSY81 + EcoRIe3 easy to aerate, yield increased 50% in fermentors

Example 38 Rescue of Plasmid DNA and Flanking DNA from MorphologicalMutants

[0607] The plasmid DNA and genomic flanking loci were isolated frommutants Aspergillus oryzae P4-8.1 and P7-14.1 using the proceduredescribed in Example 9 except for the restriction endonuclease(s) used.Transformant E. coli HB101 p4-8.1 contained a BglII rescued locus frommutant Aspergillus oryzae P4-8.1. Transformant E. coli HB101 p7-14.1contained a NarI rescued locus from mutant Aspergillus oryzae P7-14.1.

[0608] The plasmid DNA and genomic flanking loci were isolated frommutants Aspergillus oryzae DEBY7-17.2, DEBY3-2.1, DEBY5-7.1, andDEBY8-10.1. The rescued plasmids were generated as previously describedin Example 9 with the exception that rescues pSMO717, pSMO321, pHowB571,and pSMO810 were isolated from transformed E. coli DH5α cells.

[0609] Transformant E. coli DH5α pSMO717 contained the BglII rescuedlocus from mutant Aspergillus oryzae DEBY7-17.2. Transformant E. coliDH5α pSMO321 contained the BglII rescued locus from mutant Aspergilusoryzae DEBY3-2.1. Transformant E. coli DH5α pHowB571 contained the NdeIrescued locus from mutant Aspergillus oryzae DEBY5-7.1. Transformant E.coli DH5α pSMO810 contained the NdeI rescued locus from mutantAspergillus oryzae DEBY8-10.1.

Example 39 Characterization of Morphological Mutant Aspergillus oryzaeP4-8.1 Rescued Locus p4-8.1

[0610] The Aspergillus oryzae P4-8.1 rescued locus p4-8.1 containing 915and 665 bp regions on either side of the integration event was sequencedaccording to the procedure described in Example 2. The nucleic acidsequence (SEQ ID NO:50) and the deduced amino acid sequence (SEQ IDNO:51) are shown in FIG. 32. The nucleic acid sequence suggested thatthe integration event occurred within an open reading frame for ahomologue of the Saccharomyces cerevisiae YHM4 Heat Shock protein gene.The deduced amino acid sequence (SEQ ID NO:51) showed 40.2% identity tothe Saccharomyces cerevisiae YHM4 Heat Shock protein (SEQ ID NO:52) and41.8% identity to a Schizzosaccharomyces pompe Heat Shock Protein 70(SEQ ID NO:53).

Example 40 Aspergillus oryzae Transformation with BglII Linearizedp4-8.1 and Morphology Screening

[0611] To verify the link between the observed plate morphology forAspergillus oryzae P4-8.1 and the rescued genomic locus, Aspergillusoryzae HowB430 was transformed with the BglII linearized rescued locusof Aspergillus oryzae P4-8.1, p4-8.1, using the procedure described inExample 5.

[0612] Sixty-six transformants were obtained, transferred to CM-1 agar,and incubated at 34° C. for 3-4 days to evaluate the morphology. Sixteentransformants with the correct plate morphology were transferred tofresh CM-1 plates as center colonies, and 12 transformants maintainingthe plate morphology after 4 days at 34° C. were analyzed by Southernblot analysis with a PCR amplified 300 bp fragment of the rescued locusas a probe. The fragment was PCR amplified using the primers belowsynthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizeraccording to the manufacturer's instructions. HSP-1:5′-TACGGTTGACAGTGGAGC-3′ (SEQ ID NO:54) HSP-3r: 5′-CACTGACTTCTCCGATGC-3′(SEQ ID NO:55)

[0613] The amplification reaction (50 μl) contained the followingcomponents: 0.2 ng of p4-8.1, 50 pmol of primer HSP-1, 50 pmol of primerHSP-3r, 0.25 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerasebuffer, and 2.5 U of Taq polymerase. The reaction was incubated in anEricomp Thermal Cycler programmed as follows: One cycle at 95° C. for 3minutes; 30 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and72° C. for 1.5 minutes; and 1 cycle at 72° C. for 5 minutes. The PCRproduct was isolated by electrophoresis on a 1% agarose gel.

[0614] Samples of the genomic DNA were obtained from each of the 12transformants obtained according to the method described in Example 9.The genomic DNAs were digested with BglII and submitted to Southernanalysis according to the procedure described in Example 14.

[0615] All 12 transformants were affected at the rescued locus,suggesting a connection between this locus and the observed platemorphology.

Example 41 Characterization of Morphological Mutant Aspergillus oryzaeP7-14.1 Rescued Locus p7-14.1

[0616] The Aspergillus oryzae P7-14. 1 rescued locus p7-14. 1 containing1040 and 520 bp regions on either side of the integration event wassequenced according to the procedure described in Example 2. The nucleicacid sequence (SEQ ID NO:56) and the deduced amino acid sequence (SEQ IDNO:57) are shown in FIG. 33. The nucleic acid sequence suggested thatthe integration event occurred within an open reading frame for ahomologue of the Aspergillus nidulans chitin synthase B (chsB) gene andthe Aspergillus fumigatus chitin synthase G (chsG) gene. Identities of94% and 80% were found when the deduced amino acid sequences of the twosides of the rescued locus (SEQ ID NO:57), the chsB gene (SEQ ID NO:58),and the chsG gene (SEQ ID NO:59) were compared.

[0617] Disruption of the chsB gene in Aspergillus nidulans is known tochange the morphology significantly (Yanai et al., 1994, Biosci.Biotech. Biochem. 58: 1828-1835), and in Aspergillus fumigatusdisruption of the chsG gene is known to cause colonial morphology(Mellado et al., 1996, Molecular Microbiology 20: 667-679), which is theobserved phenotype of Aspergillus oryzae P7-14.1.

Example 42 Aspergillus oryzae Transformation with a Linear chs Fragmentand Morphology Screening

[0618] A 1.9 kb DNA fragment was generated by PCR using as the templateAspergillus oryzae HowB430 genomic DNA prepared as described in Example6. Primer A, 5′-CACCAAGTCAGAGCGTC-3′ (SEQ ID NO:60), was derived fromthe rescued chs Aspergillus oryzae homolog. Primer 5,5′-GGICCITTYGAYGAYCCICA-3′ (SEQ ID NO:61), was degenerate based on theconsensus sequence of the Aspergillus fumigatus chsG and Aspergillusnidulans chsB genes. The amplification reaction (50 μl) contained thefollowing components: 10 ng of pHB220, 48.4 pmol of each primer, 1 mMeach of dATP, dCTP, dGTP, and dTTP, and the Advantage-GC™ Tth PolymeraseMix (Clontech, Palo Alto, Calif.). The reaction was incubated in anEricomp Thermal Cycler programmed as follows: One cycle at 95° C. for 3minutes; 30 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and72° C. for 3 minutes; and 1 cycle at 72° C. for 5 minutes. The PCRproduct was isolated by electrophoresis on a 1% agarose gel.

[0619] The DNA fragment was cloned into the PCR-Blunt Cloning Vector(Invitrogen, San Diego, Calif.). A HindIII site in the multicloning sitewas destroyed by filling in with the Klenow fragment of DNA PolymeraseI. A 2 kb HindIII-EcoRI fragment containing the Basta gene conferringresistance to Bialaphos was obtained from pMT1612 (FIG. 34) and insertedinto the chs HindIII site located approximately 0.7 kb within the chsfragment. The resultant plasmid was labelled pHB220.

[0620] Using pHB220 as template, a 4 kb PCR fragment was generated usingprimer A and 5′-GGGCCGTTTGACAATCCGCAT-3′ (SEQ ID NO:62). Theamplification reaction was performed as described above except thereaction was incubated in an Ericomp Thermal Cycler programmed asfollows: One cycle at 95° C. for 3 minutes; 30 cycles each at 95° C. for1 minute, 58° C. for 1 minute, and 72° C. for 6 minutes; and 1 cycle at72° C. for 5 minutes. The PCR product was isolated by electrophoresis ona 1% agarose gel.

[0621] The PCR fragment was then used directly to transform protoplastsprepared from Aspergillus oryzae HowL795 according to the proceduredescribed in Example 5. Of 100 transformants, three transformantsappeared to have a “colonial” plate morphology on Minimal medium platesand PDA plates.

[0622] Southern analysis was performed on the three transformants andAspergillus oryzae HowB430 as a control using the 2 kb DIG-labelled chsfragment as probe. Genomic DNA was prepared from the three strains andcontrol strain as described in Example 9. Samples of the genomic DNAfrom each of the 3 transformants digested with HindIII was submitted toSouthern analysis according to the procedure described in Example 14.

[0623] The Southern analysis showed that each of the three transformantshad undergone a gene replacement substituting the chs/basta constructwith the wild-type chs gene. The results confirmed that the colonialmorphology observed in the chs tagged strain Aspergillus oryzae P7-14. 1wag associated with a mutation of the chs gene.

[0624] An apparent effect of the chs gene on colony morphology wasobserved in shake flask cultures containing MY25 medium performed asdescribed in Example 8. The pellet mass of the colonies in the brothappeared less dense in the chs mutants of Aspergillus oryzae HowL795compared to Aspergillus oryzae HowL795.

[0625] Fermentations were also performed as described in Example 8 ontwo derivatives of Aspergillus oryzae HowL795 containing genedisruptions of the chs gene. Lipase yields in both strains wereapproximately 21% greater than Aspergillus oryzae HowL795. The kineticsof enzyme production appeared to be increased in the chs mutants in thelater stages of fermentation suggesting that these strains exhibited amore optimal tank morphology.

Example 43 Characterization of Morphological Mutant Aspergillus oryzaeDEBY7-17.2 Rescued Locus pSMO717

[0626] The Aspergillus oryzae DEBY7-17.2 rescued locus pSMO717containing 400 bp was sequenced according to the method described inExample 2. The nucleic acid sequence (SEQ ID NO:63) and the deducedamino acid sequence (SEQ ID NO:64) are shown in FIG. 35. The deducedamino acid sequence (SEQ ID NO:64) showed 44% identity to the deducedamino acid sequence of an ORF of Aspergillus nidulans (AC000133) (SEQ IDNO:65).

Example 44 Characterization of Morphological Mutant Aspergillus oryzaeDEBY3-2.1 Rescued Locus pSMO321

[0627] The Aspergillus oryzae DEBY3-2.1 rescued locus pSMO321 containing1.0 kb was sequenced according to the method described in Example 2. Thenucleic acid sequence (SEQ ID NO:66) shown in FIG. 36 showed no homologyto any published sequences.

[0628] Probes from either end of the rescued plasmid pSMO321 weregenerated by PCR. Probe 5 was generated with primers 970850 and 970851shown below. Probe 6 was generated with primers 970852 and primer 970853shown below. The primers were synthesized with an Applied BiosystemsModel 394 DNA/RNA Synthesizer according to the manufacturer'sinstructions. The template for the 50 μl PCR labeling reaction was 10 ngof the rescued plasmid pSMO321. PCR cycles and conditions were asdescribed in Example 20. 970850: 5′-GTTCTATTGAGATACGCG-3′ (SEQ ID NO:67)970851: 5′-ACAAGCCGACCGGTTTTG-3′ (SEQ ID NO:68) 970852:5′-CGATAAGGACTCCAAGAG-3′ (SEQ ID NO:69) 970853: 5′-GTCGCGCATAATATGAAG-3′(SEQ ID NO:70)

[0629] Southern blots of Aspergillus oryzae HowB430 genomic DNA digestedwith SphI, SalI and BamH1 were prepared and analyzed according to themethod described in Example 14. The blots were hybridized independentlyto probes 5 and 6 made from the ends of the rescued plasmid.

[0630] Analysis of the Southern blots suggested no deletions hadoccurred. When PCR was performed using 500 ng of genomic DNA fromAspergillus oryzae HowB430 with primers 090850 and 090852, a 500 bpproduct was amplified as predicted verifying that no deletions had takenplace.

[0631] Genomic DNA was prepared from the tagged mutant strainAspergillus oryzae DEBY3-2.1 as described in Example 9, digested withthe restriction enzyme used for REMI (BamHI), blotted and probed withthe NheI fragment from Aspergillus oryzae pyrG. Southern analysis ofthis blot suggested the tagged plasmid had inserted into a BamHI site inthe genome.

Example 45 Characterization of Morphological Mutant Aspergillus oryzaeDEBY 5-7.1 Rescued Locus pHowB571

[0632] The Aspergillus oryzae DEBY5-7.1 rescued locus pHowB571containing 600 bp was sequenced according to the method described inExample 2. The nucleic acid sequence (SEQ ID NO:71) shown in FIG. 37showed no homology to any published sequences.

[0633] To test if a deletion occurred during tagging, a Southern blotwas preapared and analyzed according to the method described in Example14 using genomic DNA from Aspergillus oryzae HowB430 digested with SphI,SalI and BamHI. Probes from either end of the rescued tagged plasmidpSMO571 were generated by PCR. Probe 7 was generated with primer 970936and primer 970937 shown below. Probe 8 was generated with primer 970938and primer 970939 shown below. The primers were synthesized with anApplied Biosystems Model 394 DNA/RNA Synthesizer according to themanufacturer's instructions. The template for the PCR labeling reactionwas 10 ng of pSMO571. PCR cycles and conditions were as described inExample 20. 970936: 5′-CTTCCTCATAAACCACCC-3′ (SEQ ID NO:72) 970937:5′-AACTGACAGGACAAGACC-3′ (SEQ ID NO:73) 970938: 5′-GACTTGCATCACTTCCTC-3′(SEQ ID NO:74) 970939: 5′-TGAAGCTGAGAGTAGGTG-3′ (SEQ ID NO:75)

[0634] The results showed identical banding patterns from both probessuggesting no deletions had occurred. PCR was used to verify that nodeletions had occurred using the method described in Example 20. A totalof 500 ng of genomic DNA from Aspergillus oryzae HowB430 was used astemplate with primer 970936 and primer 970939. A 550 bp product wasamplified as predicted. Southern data from genomic DNA obtained from thetagged mutant Aspergillus oryzae DEBY5-7.1 digested with BamHI,hybridized to the NheI fragment of pyrG suggested that the taggedplasmid had inserted into a BamHI site in the genome. Southern blotconditions are described as above.

[0635] Southern and PCR analysis demonstrated the tagged plasmid hadinserted directly into a BamHI site. Cloning with TOPO pCR11 vector andsubsequent sequencing according to Example 2 of the PCR productgenerated using Aspergillus oryzae HowB430 genomic DNA with primers fromthe rescued ends of Aspergillus oryzae DEBY5-7.1 confirmed this result.

Example 46 Characterization of Morphological Mutant Aspergillus oryzaeDEBY8-10.1 pSMO810

[0636] The Aspergillus oryzae DEBY8-10.1 rescued locus pSMO810containing 750 bp was sequenced according to the method described inExample 2. The nucleic acid sequence (SEQ ID NO:76) shown in FIG. 38showed no homology to any published sequences.

[0637] Probes from either end of the rescued plasmid pSMO810 weregenerated by PCR. Probe 9 was generated with primer 970854 and primer970855 shown below. Probe 10 was generated with primer 970856 and primer970857 shown below. The primers were synthesized with an AppliedBiosystems Model 394 DNA/RNA Synthesizer according to the manufacturer'sinstructions. The template for the PCR labeling reaction was 10 ng ofpSMO810. The PCR reaction and conditions were as described in Example20. 970854: 5′-GTTTCGGTATTGTCACTG-3′ (SEQ ID NO:77) 970855:5′-ACAGGTGAACAACTGAGG-3′ (SEQ ID NO:78) 970856: 5′-CGACCAAACTAGACAAGC-3′(SEQ ID NO:79) 970857: 5′-CTTTCCTCTTGGACACAC-3′ (SEQ ID NO:80)

[0638] Southern analysis was performed as described in Example 14.Southern analysis of genomic DNA from, Aspergillus oryzae HowB430digested with SphI, SalI, and BamHI hybridized independently with probes9 and 10 suggested no deletions had occurred during the insertion of thetagged plasmid. PCR using 500 ng of Aspergillus oryzae HowB430 genomicDNA as template with primers 090854 and 090856 amplified a 500 bpproduct as expected with no deletions. Southern analysis of genomic DNAprepared from mutant Aspergillus oryzae DEBY8-10.1, digested with EcoRI,and probed to the NheI fragment of pyrG suggested that the plasmidintegrated into an EcoRI site in the genome. Cloning with TOPO pCR11vector and subsequent sequencing according to Example 2 of the PCRproduct generated using Aspergillus oryzae HowB430 genomic DNA withprimers from the rescued ends of Aspergillus oryzae DEBY8-10.1 confirmedthis result.

Example 47 Screening on a Poor Carbon Source for High Producers

[0639] Eighteen pools of Aspergillus oryzae HowB430 transformants, 11generated with HindIII digested pDSY81 and in the presence of HindIII,and 7 generated with SalI digested pDSY81 and in the presence of SalI(see Example 5) were screened on poor carbon sources to identify mutantswhich were high producers of lipase. Glycerol was used as poor carbonsource, since the expression of lipase is very low on glycerol, but anumber of other carbon sources, e.g., xylose, sucrose, and polyols suchas mannitol and sorbitol could be used in a similar way.

[0640] The primary 96-well plate screen was performed as described inExample 7, but with GLY25 medium composed of 100 ml of 10% yeastextract, 100 ml of 25% glycerol, 100 ml of 2% urea per liter diluted50-fold. Lipase assays were performed as described in Example 7.

[0641] Mutants of interest were then inoculated directly into 24-wellplates containing the same medium as above and grown 6 days at 34° C.and 100 rpm. Cultures were then assayed for lipase activity as describedin Example 7, and mutants of interest were plated on COVE plates toproduce spores, spread on PDA plates to produce single colonies, andthen 4 single isolates of each mutant were grown on CM-glycerol agar (asCM-1 agar, but maltose was replaced by glycerol as carbon source) toproduce spores for inoculation of 24-well plates as above.

[0642] After the 24-well plates, 10 transformants were identified forfurther evaluation in shake flasks. The shake flasks contained 50 ml ofmedium at pH 6.5 composed of 1 g of MgSO₄-7H₂O, 1 g/l K₂SO₄, 15 g ofKH2PO₄, 0.25 ml of trace metals solution, 0.7 g of yeast extract, 3 mlof 50% urea, 2 ml of 15% CaCl₂-2H₂O, and 2% carbon source (eithermaltose, glucose, sucrose, or glycerol). The shake flasks containing 50ml of medium in a 125 PP flask were inoculated with 0.5 ml G1-glyovernight culture, incubated at 34° C. and 200 rpm, and sampled after 2and 3 days. Lipase activity was measured as above.

[0643] The results are shown in Table 12 where lipase production byAspergillus oryzae HowB430 grown on glycerol as carbon source isnormalized to 1.0. TABLE 12 Strain Pool Glycerol Maltose Glucose SucroseHowB430 NA 1.0 152 44 1.1 HINL880.1 Hin-5 1.5 182 49 10 HINL895.3 Hin-208.4 140 38 11 HINL918.4 Hin-24 5.7 163 45 8.8 SALL587.4 Sal-4 1.6 178 8922 SALL591.2 Sal-4 1.3 232 126 33 SALL631.2 Sal-5 16.6 72 35 25SALL631.3 Sal-5 18.3 67 42 29 SALL664.2 Sal-15 6.1 70 79 9.2 SALL683.3Sal-16 1.6 186 77 20 SALL692.2 Sal-16 2.7 148 63 24

[0644] The relative expression of lipase responded differently todifferent carbon sources, suggesting that the regulation of the lipaseexpression was altered in these transformants.

Example 48 Screening for α-Cyclopiazonic Acid Mutants

[0645] The pools e1-e26 from the EcoRI library described in Example 5was used in screening for α-cyclopiazonic acid negative strains.

[0646] The spore number in the vials containing the different pools wasdetermined by counting an appropriate dilution in a haemocytometer and adilution series was constructed in such a way that approximately 30-50spore derived colonies were present on each 9 cm screening plate. Thescreening medium was composed per liter of 30 g of mannitol, 10 g ofglucose, 10 g of succinic acid, 3 g of Casamino acids, 1 g of KH₂PO₄,0.3 g of MgSO₄-7H₂O, 0.2 g of FeSO₄-7H₂O, 100 μl of Triton X100, and 20g of Difco Bacto Agar. The pH was adjusted to 5.6 with 14% NH₄OH beforeautoclaving. The ferrous ion forms a red complex with α-cyclopiazonicacid. This complex is seen on the reverse side of the colonies.

[0647] Approximately 2000-2500 colonies were screened from each pool.Colonies with no red coloration on the reverse side after 7 daysincubation at 34° C. were reisolated on the screening medium andincubated for 7 days at 34° C. Ten colonies originating from 6 differentpools exhibited non-red reverse coloration and were subsequentlyinoculated onto Cove-N slants made as follows: 20 ml of Cove saltsolution, 4.2 g of NaNO₃, and 60 g of glucose were dissolved indeionised water and the volume made up to 1000 ml. The medium wassolidified by 2% Difco Bacto Agar.

[0648] Five ml of a spore suspension, made from a Cove slant by adding10 ml of aqueous 0.01% Tween 80, was inoculated into 500 ml baffledshake flasks containing 100 ml of MDU1B shake flask medium. At the timeof inoculation, 1.3 ml of 50% sterile filtered urea was added to eachshake flask. The shake flasks were incubated at 250 rpm for 5 days at34° C.

[0649] Ten μl of supernatant from the 5 day old shake flask cultureswere applied to the opposite edges of a 20 cm×20 cm TLC plate (MerckSilica Gel 60). The plate was then run for 15 minutes in achloroform:acetone:propan-2-ol (85:15:20) solvent system (CAP) allowedto dry, turned around and the opposite side was run in a toluene:ethylacetate:formic acid (5:4:1) solvent system (TEF) for 15 minutes. Theplate was allowed to dry thoroughly for 1 hour in a fume hood beforespraying with Ehrlich reagent (2 g of 4-dimethylaminobenzaldehyde in 85ml of 96% ethanol plus 15 ml of 37% hydrochloric acid). α-Cyclopiazonicacid was seen as bluish-violet mushroom shaped spots with a typical lowRf value in the CAP solvent system (a neutral system) whereas the acidicTEF solvent system yielded a typical high Rf value prolonged smear.Solutions of 30, 15, and 7.5 ppm of α-cyclopiazonic acid (Sigma ChemicalCo., St. Louis, Mo.) in a 1:1:1 solution of ethanol, methanol, andchloroform were used as standards.

[0650] The TLC analysis of ten putative α-cyclopiazonic acid-freetransformants showed no sign of α-cyclopiazonic acid. The remainingcontents of the shake flasks were filtered through Miracloth and 10 mlof 0.1 M hydrochloric acid were added to 60 ml of each filtrate. Theacidified filtrates were then vigorously shaken for 3-5 minutes with 50ml chloroform. The bottom phases (approximately 25 ml) were eachtransferred to a 300 ml beaker after phase separation (3 hrs) and thechloroform allowed to evaporate. The residues were each redissolved in300 μl of chloroform and 10 μl of each concentrate was analyzed by TLCas described above.

[0651] None of the 10 strain extracts contained any detectableα-cyclopiazonic acid.

Deposit of Biological Materials

[0652] The following strains have been deposited according to theBudapest Treaty in the Agricultural Research Service Patent CultureCollection (NRRL), Northern Regional Research Laboratory, 1815University Street, Peoria, Ill. 61604, USA. Strain Accession NumberDeposit Date E. coli HB101 pDSY109 NRRL B-21623 Sep. 5, 1996 E. coliDH5α pMT1936 NRRL B-21832 Sep. 8, 1997 E. coli HB101 pDSY112 NRRLB-21622 Sep. 5, 1996 E. coli HB101 pDSY138 NRRL B-21833 Sep. 8, 1997 E.coli DH5α pDSY162 NRRL B-21831 Sep. 8, 1997 E. coli DH5α pDSY163 NRRLB-21830 Sep. 8, 1997 E. coli DH5α pSMO1204 NRRL B-21820 Sep. 8, 1997 E.coli DH5α pSMOH603 NRRL B-21821 Sep. 8, 1997 E. coli HB101 p4-8.1 NRRLB-21823 Sep. 8, 1997 E. coli HB101 p7-14.1 NRRL B-21824 Sep. 8, 1997 E.coli DH5α pHB220 NRRL B-21825 Sep. 8, 1997 E. coli DH5α pSMO717 NRRLB-21826 Sep. 8, 1997 E. coli DH5α pSMO321 NRRL B-21827 Sep. 8, 1997 E.coli DH5α pHowB571 NRRL B-21828 Sep. 8, 1997 E. coli DH5α pSMO810 NRRLB-21829 Sep. 8, 1997

[0653] The strains have been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U. S. C.§122. The deposits represent a substantially pure culture of eachdeposited strain. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

[0654] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

[0655] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the particularinformation for which the publication was cited. The publicationsdiscussed above are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

[0656] It is to be understood that this invention is not limited to theparticular methods and compositions described as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims.

[0657] Unless defined otherwise all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any materialsor methods similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are described.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:80 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATGCATCTGGAAACGCAACC CTGA 24 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 2: ATGCATTCTA CGCCAGGACC GAGC 24 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 3: TGGTGTACAG GGGCATAAAA T 21 (2) INFORMATIONFOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATTTAAATCC AGTTGTGTATATAGAGGATT GTGG 34 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 5: ATTTAAATGA TGAGGAGCTC CCTTGTGCTG 30 (2) INFORMATION FOR SEQ IDNO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 6: TTAATTAACT AGAGTCGACC CAGCCGCGC 29 (2)INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GCGGGATCCCTAGAGTAGGG GGTGGTGG 28 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 8: GCGGGATCCC CCCTAAGGAT AGGCCCTA 28 (2) INFORMATION FOR SEQ IDNO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3000 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACACGGCCTGGACAATGAGA CAACTCTTCT AAAGAGTTCA GATTTAGTAT ATATACTAGC 60 CAGGACGGACTTTCCAAATA TTATGTAAAT TAAAGTGTCG TTGTGAAGTG CTACCTATAA 120 TGCTTAGTATGTGTATGTCT GGATGCAACG GGCTAATATT ACACATCAAG AAGTCCACTC 180 AATAAGCCACTGGTGCAAAT TAACTGAATA CACATGTATC TATATCCGGA GCAATAAAAA 240 CTAACGATAATAATGGCAAG GGTCACTTAT AACAAACTCA TACTGGACAA AAAGTATGGA 300 GAAACAATAGATTAAAAAGG TCCCGTTCTA TATTTAATCC TCCCGCGACG GAGTTTCCTT 360 ATCCCGATAGATACGATAAG GATCCAGGTA ACGTCATTCC ATCCCGTGAG ATAAAGAGGA 420 CTCCACCTCAACAACTAATC ATCAAATCTC CAATCAATAT CAATGAACCC ATAACAACTT 480 AAAAAGCTCTCGGAAAAGTA AAAAGAGCTT CTATCAGCAT ATACACCATG GTCCTCGGTG 540 GATCAAGCGGGTCAAAGGTC ACTCCGTACC TGATCTACCT TGTGTTTATC ACAACTTTGG 600 GGCCACTTCAATTCGGATAT CATTTGGTAT TACACGGAGC TTGGTCTATG CTGGAGGCTT 660 CAATACATCGGCTGACAATA TATTATGATA GGCTGAGCTC AATGCCCCCC AGGCCGTGAT 720 AACTTGCGAGCGGAAAAGCA TCCATTCGAC AACAACACGG GGTCTCCCGC AATGCATACC 780 TATGAACCCATCCCAATTCG GCCTGGTCTC CTCTATATAC ACCCTTGGGG GCTTGCTAGG 840 GGCTCTCCTGGCAGGTCCAG TTTCCACCAA GCATGGCCGC TTGTTCACAC TGCGAGCGAC 900 CACCATCTTCTTCATCCTAG GCCCTATAGC AGAAACATTT GCGCCCAGTA TACCCGTATT 960 GAGTATGGGTAGGCTTTTAT CTGGTGTTGG TGCGGGCGCT TCTATCGTCG TGGGTCCGAT 1020 ATATATCTCTGAGATTGCTC CTCCTAGTGC TAAGGGTCTT TTCGGCGCTT TTACGCAAAT 1080 CATGACTAATGTCGGTATTC TGTTGACACA GTCCCTTGGT TACTTCTTGA GTAAAGGAAG 1140 TATGTGGAGAGTTATACTTG CAATTGCTGG CGCGATCGGA TGCCTTGAGC TTCTGGGCCT 1200 CTTCTTAGTCCCAGAAAGCC CCATCTGGCT TGCAGATCAC CAGAAAGGGA ATGTGGCTAG 1260 ACAGGTGCTACAACGTATAC GGGGCAGGGA TGCAGACATC GAGCCAGAGG TTGAAGGCTG 1320 GAGAACATCTGCAGCGCCTG AACACAGCTC TGGGGAAGAG CAGTCCCTAC TATCACCCCC 1380 ATCTGGAAATATGCCACCCA AGCAACCTCC GGTTACCATG ATGCGAGCTA TTACTGATTC 1440 TTTTTACCGCCCTGCCATCA TTGCAGTGGT CGGAGTCATG GTTTCCCAGC AGTTCACTGG 1500 TGTCAACAGCATCATCATGT ACAGCGTTTC CCTCTTACAG ACCATCCTTC CCACCACTGC 1560 AGCCCTGTTGTCGGTGATCA TCTCGGCTAT CAATCTTGTA ATCACTCTGG CCTGCTCACC 1620 ACTACCTGATAAGATTGGTA GACGCTCCTG CCTGCTTCTA AGTATCAGCG GCATGGGTCT 1680 TAATTCCGTCCTACTGGCGC TAGCCATCTA CTTCAACCTG AAAGCCTTAT CCGCCATAGC 1740 AGTTCTACTTTTCGTTGCTT CTTTCGCCGC CGGTCTAGGC CCAGTCCCCT TCATTTTAGC 1800 CTCTGAACTCGTTGGCCCGG AGGCTGTCGG CGCCGCACAG AGCTGGGCGC TGGGAGCGAA 1860 CTGGATTGCCACGTTCATCG TGGCACAATT TTTTCCGATG TTAAACGATT TGTTGGGCGG 1920 ACGAGGCAAGATCTACTGGA TCTTTGCAGC GATGGCCTGT CTCCTCGGAA GTTTCATCTA 1980 CTGGTGGGTGCCGGAGACCA AGGGGAAGGC TAACGCCGAC GAAGTTTGGG GAAGGACCAA 2040 CCAGCGCAGACAGGACTAAT TTTTCTGGCC TCTTTGATTT TTTTTTTCTG GGCCTTACTC 2100 TGCTGCCAACATTCAGATTA TCAATTAGTA GTCAATCTGT GACTATCCTC TCCGAGGGAT 2160 AGCTTGCAAAGGTGTGACCT CCACAGAGGA ATCTATCGTG TGACAGTATC AAAGACAATA 2220 GAATAGCAATAATTGGTGCT CTCTACCTAG GAGCATTCGG TGAGAGTGAA AGAGTCATAC 2280 TTGCCTCGGCTTGTTCATCC CAGTCGATCA GTCAGGTTTA GCTCGGCAGT AAAAGCAATA 2340 CCGGTCTACTTCCATCTTCA AACTGTACCG CGGAAACAAA GAGTAAAGGA GGGGTCATGA 2400 TACCTCTAAATAATGTATAA GTCGTTGACA ATGCTCTTTA TCACCACCCG TTGAAGACGT 2460 CCTTTGATGTCTTGATCATC ACAAGCAGGT TGATCATCTG CGATCGACGT CACTTCGCAC 2520 CGCACACTGCATGACAAGTG CGGGGCAGAG GGGCCAACAG GCCCAAAAAT TTAGTGATTT 2580 TGAAGCAATGTTGTTACACC CTTTTACCCC TCAATCCATG ATAAGGGAAA AAGAGATGCT 2640 GAGAGAGGGTCACTGCCACG CTAGACTGGA TTGGTCCGTA TATGCAGGTT TATGCACGCA 2700 CAGGGGGGCTTCGTTCTTCT TGGCTATGCA CTATGGATTA GTAGGGTGTA TTCAACCACG 2760 TAGATAGATTGGCGTGTCCG GTGCAGGATA TGTAGAAGAC AATGAGGTTC GGGCTTTCGG 2820 AAATGAGGAAAGAATGTTGG ACAGATGAAA AACGGTACTG CTGTTGCAAG GGGGCGCTGT 2880 TTGAGATATTTTAAGTGCCT GTCATGTAAT TTTGCAACGG TGAGACATTT ATCTAGGGTA 2940 AAATCCAAGAAGAACCTAGG GAAGAGTAAA GCCACAACGA AGATTACGTG AGAGGAAGAG 3000 (2)INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:488 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 10: Met Val Leu Gly Gly Ser Ser Gly Ser Lys Val Thr Pro Tyr LeuIle 1 5 10 15 Tyr Leu Val Phe Ile Thr Thr Leu Gly Pro Leu Gln Phe GlyTyr His 20 25 30 Leu Ala Glu Leu Asn Ala Pro Gln Ala Val Ile Thr Cys GluArg Lys 35 40 45 Ser Ile His Ser Thr Thr Thr Arg Gly Leu Pro Gln Cys IlePro Met 50 55 60 Asn Pro Ser Gln Phe Gly Leu Val Ser Ser Ile Tyr Thr LeuGly Gly 65 70 75 80 Leu Leu Gly Ala Leu Leu Ala Gly Pro Val Ser Thr LysHis Gly Arg 85 90 95 Leu Phe Thr Leu Arg Ala Thr Thr Ile Phe Phe Ile LeuGly Pro Ile 100 105 110 Ala Glu Thr Phe Ala Pro Ser Ile Pro Val Leu SerMet Gly Arg Leu 115 120 125 Leu Ser Gly Val Gly Ala Gly Ala Ser Ile ValVal Gly Pro Ile Tyr 130 135 140 Ile Ser Glu Ile Ala Pro Pro Ser Ala LysGly Leu Phe Gly Ala Phe 145 150 155 160 Thr Gln Ile Met Thr Asn Val GlyIle Leu Leu Thr Gln Ser Leu Gly 165 170 175 Tyr Phe Leu Ser Lys Gly SerMet Trp Arg Val Ile Leu Ala Ile Ala 180 185 190 Gly Ala Ile Gly Cys LeuGlu Leu Leu Gly Leu Phe Leu Val Pro Glu 195 200 205 Ser Pro Ile Trp LeuAla Asp His Gln Lys Gly Asn Val Ala Arg Gln 210 215 220 Val Leu Gln ArgIle Arg Gly Arg Asp Ala Asp Ile Glu Pro Glu Val 225 230 235 240 Glu GlyTrp Arg Thr Ser Ala Ala Pro Glu His Ser Ser Gly Glu Glu 245 250 255 GlnSer Leu Leu Ser Pro Pro Ser Gly Asn Met Pro Pro Lys Gln Pro 260 265 270Pro Val Thr Met Met Arg Ala Ile Thr Asp Ser Phe Tyr Arg Pro Ala 275 280285 Ile Ile Ala Val Val Gly Val Met Val Ser Gln Gln Phe Thr Gly Val 290295 300 Asn Ser Ile Ile Met Tyr Ser Val Ser Leu Leu Gln Thr Ile Leu Pro305 310 315 320 Thr Thr Ala Ala Leu Leu Ser Val Ile Ile Ser Ala Ile AsnLeu Val 325 330 335 Ile Thr Leu Ala Cys Ser Pro Leu Pro Asp Lys Ile GlyArg Arg Ser 340 345 350 Cys Leu Leu Leu Ser Ile Ser Gly Met Gly Leu AsnSer Val Leu Leu 355 360 365 Ala Leu Ala Ile Tyr Phe Asn Leu Lys Ala LeuSer Ala Ile Ala Val 370 375 380 Leu Leu Phe Val Ala Ser Phe Ala Ala GlyLeu Gly Pro Val Pro Phe 385 390 395 400 Ile Leu Ala Ser Glu Leu Val GlyPro Glu Ala Val Gly Ala Ala Gln 405 410 415 Ser Trp Ala Leu Gly Ala AsnTrp Ile Ala Thr Phe Ile Val Ala Gln 420 425 430 Phe Phe Pro Met Leu AsnAsp Leu Leu Gly Gly Arg Gly Lys Ile Tyr 435 440 445 Trp Ile Phe Ala AlaMet Ala Cys Leu Leu Gly Ser Phe Ile Tyr Trp 450 455 460 Trp Val Pro GluThr Lys Gly Lys Ala Asn Ala Asp Glu Val Trp Gly 465 470 475 480 Arg ThrAsn Gln Arg Arg Gln Asp 485 (2) INFORMATION FOR SEQ ID NO: 11: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 488 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Ala Glu Thr Glu ArgLeu Met Pro Asn Gly Gly Ser Arg Glu Thr 1 5 10 15 Lys Pro Leu Ile ThrGly His Leu Ile Leu Gly Thr Ile Val Ala Cys 20 25 30 Leu Gly Ser Ile GlnTyr Gly Tyr His Ile Ala Glu Leu Asn Ala Pro 35 40 45 Gln Glu Phe Leu SerCys Ser Arg Phe Glu Ala Pro Asp Glu Asn Ile 50 55 60 Ser Tyr Asp Asp ThrTrp Val Gly Gln His Gly Leu Lys Gln Cys Ile 65 70 75 80 Ala Leu Thr AspSer Gln Tyr Gly Ala Ile Thr Ser Ile Phe Ser Ile 85 90 95 Gly Gly Leu PheGly Ser Tyr Tyr Ala Gly Asn Trp Ala Asn Arg Tyr 100 105 110 Gly Arg LysTyr Val Ser Met Gly Ala Ser Ala Met Cys Met Val Ser 115 120 125 Ser LeuLeu Leu Phe Phe Ser Asn Ser Tyr Leu Gln Leu Leu Phe Gly 130 135 140 ArgPhe Leu Val Gly Met Ser Cys Gly Thr Ala Ile Val Ile Thr Pro 145 150 155160 Leu Phe Ile Asn Glu Ile Ala Pro Val Glu Trp Arg Gly Ala Met Gly 165170 175 Ser Met Asn Gln Val Ser Ile Asn Leu Gly Ile Leu Leu Thr Gln Thr180 185 190 Leu Ala Leu Lys Tyr Ala Asp Ser Tyr Asn Trp Arg Trp Leu LeuPhe 195 200 205 Ser Gly Ser Val Ile Ala Val Ala Asn Ile Leu Ala Trp LeuLys Val 210 215 220 Asp Glu Ser Pro Arg Trp Leu Val Ser His Gly Phe ValSer Glu Ala 225 230 235 240 Glu Thr Ala Leu Phe Lys Leu Arg Pro Gly ThrTyr Gln Gln Ala Lys 245 250 255 Gln Glu Ile Gln Asp Trp Gln Arg Ser HisGly His Asn Arg Asp Pro 260 265 270 Glu Ser Ser Glu Glu Thr His Ser GlyPro Thr Leu Trp Gln Tyr Val 275 280 285 Thr Asp Pro Ser Tyr Lys Lys ProArg Thr Val Ile Leu Ala Ile Leu 290 295 300 Ser Cys Gln Gln Phe Cys GlyIle Asn Ser Ile Ile Phe Tyr Gly Val 305 310 315 320 Lys Val Ile Gly LysIle Leu Pro Asp Tyr Ser Ile Gln Val Asn Phe 325 330 335 Ala Ile Ser IleLeu Asn Val Val Val Thr Leu Ala Ala Ser Ala Ile 340 345 350 Ile Asp HisVal Gly Arg Arg Pro Leu Leu Leu Ala Ser Thr Thr Val 355 360 365 Met ThrAla Met Ser Leu Leu Ile Ser Val Gly Leu Thr Leu Ser Val 370 375 380 SerPhe Leu Leu Val Thr Ala Thr Phe Val Tyr Ile Ala Ala Phe Ala 385 390 395400 Ile Gly Leu Gly Pro Ile Pro Phe Leu Ile Ile Gly Glu Leu Ser Tyr 405410 415 Pro Gln Asp Ala Ala Thr Ala Gln Ser Phe Gly Thr Val Cys Asn Trp420 425 430 Leu Ala Thr Phe Ile Val Gly Tyr Leu Phe Pro Ile Gly His GlyLeu 435 440 445 Met Gly Gly Tyr Val Phe Ala Ile Phe Ala Ala Ile Ala AlaMet Phe 450 455 460 Ala Thr Tyr Val Tyr Lys Arg Val Pro Glu Thr Lys GlyLys Thr Thr 465 470 475 480 Tyr Ser Glu Val Trp Ala Gly Tyr 485 (2)INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:524 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 12: Met Thr Glu Asp Lys Val Thr Gly Thr Leu Val Phe Thr Val IleThr 1 5 10 15 Ala Val Leu Gly Ser Phe Gln Phe Gly Tyr Asp Ile Gly ValIle Asn 20 25 30 Ala Pro Gln Gln Val Ile Ile Ser His Tyr Arg His Val LeuGly Val 35 40 45 Pro Leu Asp Asp Arg Lys Ala Ile Asn Asn Tyr Val Ile AsnSer Thr 50 55 60 Asp Glu Leu Pro Thr Ile Ser Tyr Ser Met Asn Pro Lys ProThr Pro 65 70 75 80 Trp Ala Glu Glu Glu Thr Val Ala Ala Ala Gln Leu IleThr Met Leu 85 90 95 Trp Ser Leu Ser Val Ser Ser Phe Ala Val Gly Gly MetThr Ala Ser 100 105 110 Phe Phe Gly Gly Trp Leu Gly Asp Thr Leu Gly ArgIle Lys Ala Met 115 120 125 Leu Val Ala Asn Ile Leu Ser Leu Val Gly AlaLeu Leu Met Gly Phe 130 135 140 Ser Lys Leu Gly Pro Ser His Ile Leu IleIle Ala Gly Arg Ser Ile 145 150 155 160 Ser Gly Leu Tyr Cys Gly Leu IleSer Gly Leu Val Pro Met Tyr Ile 165 170 175 Gly Glu Ile Ala Pro Thr AlaLeu Arg Gly Ala Leu Gly Thr Phe His 180 185 190 Gln Leu Ala Ile Val ThrGly Ile Leu Ile Ser Gln Ile Ile Gly Leu 195 200 205 Glu Phe Ile Leu GlyAsn Tyr Asp Leu Trp His Ile Leu Leu Gly Leu 210 215 220 Ser Gly Val ArgAla Ile Leu Gln Ser Leu Leu Leu Phe Phe Cys Pro 225 230 235 240 Glu SerPro Arg Tyr Leu Tyr Ile Lys Leu Asp Glu Glu Val Lys Ala 245 250 255 LysGln Ser Leu Lys Arg Leu Arg Gly Tyr Asp Asp Val Thr Lys Asp 260 265 270Ile Asn Glu Met Arg Lys Glu Arg Glu Glu Ala Ser Ser Glu Gln Lys 275 280285 Val Ser Ile Ile Gln Leu Phe Thr Asn Ser Ser Tyr Arg Gln Pro Ile 290295 300 Leu Val Ala Leu Met Leu His Val Ala Gln Gln Phe Ser Gly Ile Asn305 310 315 320 Gly Ile Phe Tyr Tyr Ser Thr Ser Ile Phe Gln Thr Ala GlyIle Ser 325 330 335 Lys Pro Val Tyr Ala Thr Ile Gly Val Gly Ala Val AsnMet Val Phe 340 345 350 Thr Ala Val Ser Val Phe Leu Val Glu Lys Ala GlyArg Arg Ser Leu 355 360 365 Phe Leu Ile Gly Met Ser Gly Met Phe Val CysAla Ile Phe Met Ser 370 375 380 Val Gly Leu Val Leu Leu Asn Lys Phe SerTrp Met Ser Tyr Val Ser 385 390 395 400 Met Ile Ala Ile Phe Leu Phe ValSer Phe Phe Glu Ile Gly Pro Gly 405 410 415 Pro Ile Pro Trp Phe Met ValAla Glu Phe Phe Ser Gln Gly Pro Arg 420 425 430 Pro Ala Ala Leu Ala IleAla Ala Phe Ser Asn Trp Thr Cys Asn Phe 435 440 445 Ile Val Ala Leu CysPhe Gln Tyr Ile Ala Asp Phe Cys Gly Pro Tyr 450 455 460 Val Phe Phe LeuPhe Ala Gly Val Leu Leu Ala Phe Thr Leu Phe Thr 465 470 475 480 Phe PheLys Val Pro Glu Thr Lys Gly Lys Ser Phe Glu Glu Ile Ala 485 490 495 AlaGlu Phe Gln Lys Lys Ser Gly Ser Ala His Arg Pro Lys Ala Ala 500 505 510Val Glu Met Lys Phe Leu Gly Ala Thr Glu Thr Val 515 520 (2) INFORMATIONFOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 584 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: MetGly Ile His Ile Pro Tyr Leu Thr Ser Lys Thr Ser Gln Ser Asn 1 5 10 15Val Gly Asp Ala Val Gly Asn Ala Asp Ser Val Glu Phe Asn Ser Glu 20 25 30His Asp Ser Pro Ser Lys Arg Gly Lys Ile His Ile Glu Ser His Glu 35 40 45Ile Gln Arg Ala Pro Ala Ser Asp Asp Glu Asp Arg Ile Gln Ile Lys 50 55 60Pro Val Asn Asp Glu Asp Asp Thr Ser Val Met Ile Thr Phe Asn Gln 65 70 7580 Ser Leu Ser Pro Phe Ile Ile Thr Leu Thr Phe Val Ala Ser Ile Ser 85 9095 Gly Phe Met Phe Gly Tyr Asp Thr Gly Tyr Ile Ser Ser Ala Leu Ile 100105 110 Ser Ile Gly Thr Asp Leu Asp His Lys Val Leu Thr Tyr Gly Glu Lys115 120 125 Glu Ile Val Thr Ala Ala Thr Ser Leu Gly Ala Leu Ile Thr SerIle 130 135 140 Phe Ala Gly Thr Ala Ala Asp Ile Phe Gly Arg Lys Arg CysLeu Met 145 150 155 160 Gly Ser Asn Leu Met Phe Val Ile Gly Ala Ile LeuGln Val Ser Ala 165 170 175 His Thr Phe Trp Gln Met Ala Val Gly Arg LeuIle Met Gly Phe Gly 180 185 190 Val Gly Ile Gly Ser Leu Ile Ala Pro LeuPhe Ile Ser Glu Ile Ala 195 200 205 Pro Lys Met Ile Arg Gly Arg Leu ThrVal Ile Asn Ser Leu Trp Leu 210 215 220 Thr Gly Gly Gln Leu Val Ala TyrGly Cys Gly Ala Gly Leu Asn Tyr 225 230 235 240 Val Asn Asn Gly Trp ArgIle Leu Val Gly Leu Ser Leu Ile Pro Thr 245 250 255 Ala Val Gln Phe ThrCys Leu Cys Phe Leu Pro Asp Thr Pro Arg Tyr 260 265 270 Tyr Val Met LysGly Asp Leu Ala Arg Ala Thr Glu Val Leu Lys Arg 275 280 285 Ser Tyr ThrAsp Thr Ser Glu Glu Ile Ile Glu Arg Lys Val Glu Glu 290 295 300 Leu ValThr Leu Asn Gln Ser Ile Pro Gly Lys Asn Val Pro Glu Lys 305 310 315 320Val Trp Asn Thr Ile Lys Glu Leu His Thr Val Pro Ser Asn Leu Arg 325 330335 Ala Leu Ile Ile Gly Cys Gly Leu Gln Ala Ile Gln Gln Phe Thr Gly 340345 350 Trp Asn Ser Leu Met Tyr Phe Ser Gly Thr Ile Phe Glu Thr Val Gly355 360 365 Phe Lys Asn Ser Ser Ala Val Ser Ile Ile Val Ser Gly Thr AsnPhe 370 375 380 Ile Phe Thr Leu Val Ala Phe Phe Ser Ile Asp Lys Ile GlyArg Arg 385 390 395 400 Thr Ile Leu Leu Ile Gly Leu Pro Gly Met Thr MetAla Leu Val Val 405 410 415 Cys Ser Ile Ala Phe His Phe Leu Gly Ile LysPhe Asp Gly Ala Val 420 425 430 Ala Val Val Val Ser Ser Gly Phe Ser SerTrp Gly Ile Val Ile Ile 435 440 445 Val Phe Ile Ile Val Phe Ala Ala PheTyr Ala Leu Gly Ile Gly Thr 450 455 460 Val Pro Trp Gln Gln Ser Glu LeuPhe Pro Gln Asn Val Arg Gly Ile 465 470 475 480 Gly Thr Ser Tyr Ala ThrAla Thr Asn Trp Ala Gly Ser Leu Val Ile 485 490 495 Ala Ser Thr Phe LeuThr Met Leu Gln Asn Ile Thr Pro Ala Gly Thr 500 505 510 Phe Ala Phe PheAla Gly Leu Ser Cys Leu Ser Thr Ile Phe Cys Tyr 515 520 525 Phe Cys TyrPro Glu Leu Ser Gly Leu Glu Leu Glu Glu Val Gln Thr 530 535 540 Ile LeuLys Asp Gly Phe Asn Ile Lys Ala Ser Lys Ala Leu Ala Lys 545 550 555 560Lys Arg Lys Gln Gln Val Ala Arg Val His Glu Leu Lys Tyr Glu Pro 565 570575 Thr Gln Glu Ile Ile Glu Asp Ile 580 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 14: CTGCCGTCGA AGGTGTCCAA G 21 (2) INFORMATIONFOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ATTGTGGCCC CTATGTGGAT T21 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 4700 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 16: ACAGCGACTG GGATGGTGAA TATCTGAGCG ATAGCCCCCG ACACAGCACCAAGGGTAAGC 60 TCCATAGCGG TTCCAGGTGG CTTAGATACG CTCTTCGATG CCATATAAAGGCTTCTAACC 120 ACGCTGTACC AGTAGAAGTA CGCAAAGTTG GTCGAGGCCA CGCCAAGCAAAGAACCAACC 180 ATCCCGGAAT ATAAACCTTC AATTCCCTCT TTCTCCACAA TCTTGTTGATGGCATCTAGG 240 GTCGACTCGT AATGTACTAC ATCTCCGCTT TTCGATTCAG GTGCGTTCTTCACTTGGACT 300 TGAAGTTTGG TTTTGACACT AAAGATTAGG AACGAGTCAG TCTCAGTCTACACCACTAAG 360 CCAACTGGCA AGGCTATGTA CGCACAGGTC CAGTGGATAG ACGATAGCATTTGCGAGAAC 420 AGCACCAGTT GCACCTGCGA CAGCACTACC CCAAGGGGAG AGCGCGGGTTTCGATTGGCC 480 GGCCATTATG CAGGATGAGC TAAAGTGCCT CTGCCAATTC CGTCAGAAAGAATAGTATAA 540 GAGCACAAAT ACTGGTAAAA CCAAGACCGG CGAATGAGGC AGGACTCTGTGCGATTCGGG 600 GGGTTTAGCG TTCGGCTTGA AGGCTTACCG GATCGACTGA TAGAAAAGTTGAATGCCGAG 660 TAAGGTGAAA AGACCCCAGC TCCCAAGCAG CAACAGTAAG TGGAAGAGCTTAAGGATAGA 720 AAAAATAAAT TAGGATTAAG AAAAAAAGAA GAACCTCAAG ACTGGTCACACAGTCCCGGC 780 ATCCTGAACG TAAAATGCGG GAAGGATAGA GTCGGCAGGG CCAGGGCAGTTGCACCTCGG 840 CGCTCTGGTT TGCGCATGAC GAAATGAGCC GAGGTTCGTT TTTTGGAGGCCAATTTCTGA 900 ACACCGACCT TCGAATTCCC GTTCCTCCCC ACCGACACGC TAGTGAATGATCCAGCAAGC 960 ATACTTGGTG TTGTTTTGAC CTCATTCCAC TGCGTGTGAA TTAGCATTAATTTAAGTTTA 1020 TGATTAACAG TCAATTGCTA TACGCGAAAA TCATCATCGT CTTGATTGGCCCTTCATAAA 1080 ACTTGACAAG GAAGTTTGAT CGACCTCGGA TGTCGCGCTT TCGGAAATTTCACGGAGCCC 1140 TTCGGACGGG TCACAAGCAA GGTGTCTGAC TGTCTCGTTT AGTCGGATAGACGCTAGTTG 1200 AACTGTTATG CCTATCGCGG GGAAGATCTC GGAGTGTCAC GGTGTTTGAAGATCCCAGGC 1260 GCTCGTCAAA ATACTGCCCG GCCTGCCAGT ATGTCTAGAC CGAACGCCTCAGCCCAGAAG 1320 TCCTTTATAA CTCAGGCACT GGTACTTGAC CCTTTTTTTT TATGGTTTTTTGTTTCTTTC 1380 TTGTTACACC TTATTTTTCT TCTTCTCGTT TTTTGTAGAT AATACTGACCACTGGCTAGA 1440 AAGCCGAGCG GGATGTATCG TCCGCCACTT CTCAAAGGCA AGCTTTAGAAGCTGCCATTG 1500 ATGCTGCTGA ACACTATATG AAAGCCTTAA ATCTGGCATC TGTTCAGAAAGACAAACATG 1560 CATTGGATGC AAAGTGTAAA GAATGGCTCA CAAGAGCGGA AAAGATCAAAGAATCTAAGG 1620 ACTGGCAAGC TGCTGCCCGT TTCCATGACA AAACTGTTCC AGAGCCACGGTTGCCTGTAT 1680 CTACTCGTAA GCTCACCACA CGGGAGGAGA TCATTCTGCT AGAGGGAGCCAAGTTGAATG 1740 GCTTCATATT CCCTCCATGG TCCACCTCCC CAGGCTCTGA CGAGTTCAAACGAGAGGATG 1800 GTGAATCCCC GTTTACGTAA GTTCTGGTGG TCTGCATCGT CAATGTTGCATGTATACCCA 1860 GATGACTGCT GGATATTCTA ACCGATAACA GCGACAAACC CGATCTTCATCTATCTTATC 1920 CTCAAAGGAA AGTTTTTGAT GGCTGGAAAC GACCTTCCGA GCTTCTCGCGAAAGACACGG 1980 AAGATGTGTA CACAAAGGTG GTTCCTGTGA TGTCTGTTCC AGGAAAGACAGATCTAGTCC 2040 AGGATATGCT GACGGACTGT TCTGTCGTTG CTAGCCTTTG TGCTACTACGTCAATGCTAG 2100 AACGCGGCCA GTGTACTGTA AGAAGATTGA TCCCTTCCGG CTGACCTGCATGGTTCGCTG 2160 TGACTAATAG GTGTAGCATT TTCTTCCAAT GATATACCCT AGCCGGGGGAGCTCTCAGCC 2220 TTCACCGTCA GGCAAGTATA TATTTCGCTT TTATTTCAAT GGGTGCTTCCGGAAAGTCAT 2280 CATTGACGAC CGTTTGCCAT CGTCTAAGAC ATCAAGATCA CTCCACGTGATCGACCGGAA 2340 AAATCCCAAT TTCCTTTGGC CGGCGCTCGT AGAGAAGGCG TATTTGAAATTGCGCGGAGG 2400 CTATGATTTT CCCGGAAGCA ATTCCGGGAC AGATCTCTGG GTGCTGACAGGTTGGATTCC 2460 CGAGCAAGTC TTTCTCCATA ATGACGATGT GACTGGCGAC CAGCTCTGGAAGCGACTTTA 2520 CAGATCCTTT CACCAAGGAG ATGTTCTCTT GACTATAGGT ACCGGTGAACTCACTGAGAG 2580 GGAACAAAGA GAACTAGGCC TCGTGAGTGA GCATGATTAT GCTATTCTGGATATGAAGGA 2640 ATCTAAAGGT CGCCGACAAT TACTCGTGAA AAACCCTTGG GCTGGAGCAGATACTGCCCC 2700 CGGCGACAAT GGAAGCCTCT CTGCATCGCA GGATTTACCC CATAACCCGCCCTCATTTGA 2760 GCCGGGTACC TTTTGGATGG ATTGCGAAAA GCTGCTTCAA CATTTTGAAAACCTCTATTT 2820 GAATTGGAAC CCTGAGATTT TCAAATACCG CGAAGACGTC CACTTTACGTGGGACCTCAA 2880 CAACGGGAGA GGTGTAGCCG GCTGTTTTGT GAATAACCCG CAGTTCGCAGTGTCAACCGA 2940 GAACGGTGGG ATTGTCTGGT TACTTCTAGG CAAGCATTTC AGAACAACAGGGCAGCCGGA 3000 ACGACCTCTT GACGAATACC AAGCGAATGA GGAGTCGGCT TTTATAAGCATATATGTCTT 3060 TAACGCAGAT GGCAAACGGG TCTCTTTGAG TGATGGGGCT CTACATCGTGGCCCCTATGT 3120 GGATTCCCCT AATACGCTCA TGAGGTTAGA GATGCCCCCC AGAACAACATACACAGTCGT 3180 GGTCTCCGAG CAATCACTGC CATCTTTGAA TCAAAACTTT ACTTTGTCTGCCTTCTCTAC 3240 CTGCCCTGTA CGGATGGCAA AAGCCCAAGA TAAATACATG TGTGTCAGGAAGATTCAAGG 3300 GTCTTGGACA CCTTCGACGG CAGGTGGGAA TGCCGAATCT TCTCGATATCCACTCAACCC 3360 CCAATTTAGG TTGGAGATAG AGAATGACAC AGATGTTTCA CTCCTGCTGGAATGCCCAAA 3420 CACGGAACTC GCGACCCATG TTAAGTTATT CTGGTCCAAT GGAAATCGTGTGTCGCGAGT 3480 ACGCAGTCGC GACATAATCG CTGATAGTGG TGACTATCGC CGTGGTGGCTCCCTTGTGGA 3540 AAAGAAGGCT CTGGAACCGG GCTCATATAC AATCGTCTGT TCCACATTCGCGCCGGATCA 3600 ACTTGGCCGA TTCACGCTCT GGGTATCCTC CTTAGTTCCT TGCAAGACGAGCCCGCTCCC 3660 ACCAGAGGCA GCAGGTCGAC GAACGGTCAT TTCAGATATT GGCGTACTGCCTCCCGGGAG 3720 AGACCGAATG TTAGCTTCTC TGCAAGTGCC GCGGCTTACG AGGATCAAGCTCATCACCCG 3780 AAGTAGGCAA TCCATCATCG GGAGCCATCC TGTTGGACCC TCGCCCGTTTTAATGACAGT 3840 GGAGCTCGGG CAAGGGCCAT ACAAACAGAT CCTGGCGACT TCGGAAGATGGAACTCACAG 3900 TGATGCTGTA TCGGGGGTAC GTGTTGAGGA CTTTGACTTG CAGCCTGGGCTAGAGGAGAG 3960 TGGTGGTATT TGGATTGTTA TTGAGAGGAT TGGGGGTCCT GGAGGGCAGGTAGAGGACCA 4020 CTTTGAGGTG GAAGCTTTGG CTGAAGAGAG GGTTGAGATT GGGGAGTGGATACTTGAAGA 4080 TGCTTGATCT CTTATCCTGC AGAAGCTCTG AAGCTCTGGA CGGTCTTAGTTGAGCTTTTT 4140 TGATCGTCGT TGTGATTAGC ACGTTAGAGT AGAAGAGCGG AAACAATGATAGACATGAAT 4200 TTCTCTTATT GTCTCTATTG GCCAGAAGAG AAAGAAGGCA TTAAATCAATACATTAAAAG 4260 CAAGAGTTTA TCTATAGATA CCGAGCAGCC TCAGGATTTG AGCTGAGGTTTGTCGCGATC 4320 GCGACCGCCA AAATGGAGTT AGCTTGCTTT ACTCCGCATA AATTAAATCCTCGGCTCGGG 4380 GCCCGAATTC CCCTCCCGAG TGCTTTCAAC GTCATCCCGT TTGCTGTGTGCATTGTGCCT 4440 CCCCACCTTT AACAATTGGA GCTCTGTCCA AGGACAAATC CTTATTCTCGCGGCCTCCAT 4500 GGCGACTAAT ATTCCTGCTG CTCTGAAGTC TGCGGACATT GGGCGCTTTGCCGTCAGAGC 4560 AGCTCAGCTT GAACGTGTAA AGCCCGTGGT CGCCTACTGG TGTGAGTATTGTGTGATTAT 4620 ACCCAGTACG AACTACGAGC TGATGAGCGG CCTCTGCTGA TCGCAGGCAACTTCTGGATC 4680 GTCAACCAGA TTATTGAGAA 4700 (2) INFORMATION FOR SEQ IDNO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 854 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Met SerArg Pro Asn Ala Ser Ala Gln Lys Ser Phe Ile Thr Gln Ala 1 5 10 15 LeuLys Ala Glu Arg Asp Val Ser Ser Ala Thr Ser Gln Arg Gln Ala 20 25 30 LeuGlu Ala Ala Ile Asp Ala Ala Glu His Tyr Met Lys Ala Leu Asn 35 40 45 LeuAla Ser Val Gln Lys Asp Lys His Ala Leu Asp Ala Lys Cys Lys 50 55 60 GluTrp Leu Thr Arg Ala Glu Lys Ile Lys Glu Ser Lys Asp Trp Gln 65 70 75 80Ala Ala Ala Arg Phe His Asp Lys Thr Val Pro Glu Pro Arg Leu Pro 85 90 95Val Ser Thr Arg Lys Leu Thr Thr Arg Glu Glu Ile Ile Leu Leu Glu 100 105110 Gly Ala Lys Leu Asn Gly Phe Ile Phe Pro Pro Trp Ser Thr Ser Pro 115120 125 Gly Ser Asp Glu Phe Lys Arg Glu Asp Gly Glu Ser Pro Phe Thr Asp130 135 140 Lys Pro Asp Leu His Leu Ser Tyr Pro Gln Arg Lys Val Phe AspGly 145 150 155 160 Trp Lys Arg Pro Ser Glu Leu Leu Ala Lys Asp Thr GluAsp Val Tyr 165 170 175 Thr Lys Val Val Pro Val Met Ser Val Pro Gly LysThr Asp Leu Val 180 185 190 Gln Asp Met Leu Thr Asp Cys Ser Val Val AlaSer Leu Cys Ala Thr 195 200 205 Thr Ser Met Leu Glu Arg Gly Gln Cys ThrHis Phe Leu Pro Met Ile 210 215 220 Tyr Pro Ser Arg Gly Ser Ser Gln ProSer Pro Ser Gly Lys Tyr Ile 225 230 235 240 Phe Arg Phe Tyr Phe Asn GlyCys Phe Arg Lys Val Ile Ile Asp Asp 245 250 255 Arg Leu Pro Ser Ser LysThr Ser Arg Ser Leu His Val Ile Asp Arg 260 265 270 Lys Asn Pro Asn PheLeu Trp Pro Ala Leu Val Glu Lys Ala Tyr Leu 275 280 285 Lys Leu Arg GlyGly Tyr Asp Phe Pro Gly Ser Asn Ser Gly Thr Asp 290 295 300 Leu Trp ValLeu Thr Gly Trp Ile Pro Glu Gln Val Phe Leu His Asn 305 310 315 320 AspAsp Val Thr Gly Asp Gln Leu Trp Lys Arg Leu Tyr Arg Ser Phe 325 330 335His Gln Gly Asp Val Leu Leu Thr Ile Gly Thr Gly Glu Leu Thr Glu 340 345350 Arg Glu Gln Arg Glu Leu Gly Leu Val Ser Glu His Asp Tyr Ala Ile 355360 365 Leu Asp Met Lys Glu Ser Lys Gly Arg Arg Gln Leu Leu Val Lys Asn370 375 380 Pro Trp Ala Gly Ala Asp Thr Ala Pro Gly Asp Asn Gly Ser LeuSer 385 390 395 400 Ala Ser Gln Asp Leu Pro His Asn Pro Pro Ser Phe GluPro Gly Thr 405 410 415 Phe Trp Met Asp Cys Glu Lys Leu Leu Gln His PheGlu Asn Leu Tyr 420 425 430 Leu Asn Trp Asn Pro Glu Ile Phe Lys Tyr ArgGlu Asp Val His Phe 435 440 445 Thr Trp Asp Leu Asn Asn Gly Arg Gly ValAla Gly Cys Phe Val Asn 450 455 460 Asn Pro Gln Phe Ala Val Ser Thr GluAsn Gly Gly Ile Val Trp Leu 465 470 475 480 Leu Leu Gly Lys His Phe ArgThr Thr Gly Gln Pro Glu Arg Pro Leu 485 490 495 Asp Glu Tyr Gln Ala AsnGlu Glu Ser Ala Phe Ile Ser Ile Tyr Val 500 505 510 Phe Asn Ala Asp GlyLys Arg Val Ser Leu Ser Asp Gly Ala Leu His 515 520 525 Arg Gly Pro TyrVal Asp Ser Pro Asn Thr Leu Met Arg Leu Glu Met 530 535 540 Pro Pro ArgThr Thr Tyr Thr Val Val Val Ser Glu Gln Ser Leu Pro 545 550 555 560 SerLeu Asn Gln Asn Phe Thr Leu Ser Ala Phe Ser Thr Cys Pro Val 565 570 575Arg Met Ala Lys Ala Gln Asp Lys Tyr Met Cys Val Arg Lys Ile Gln 580 585590 Gly Ser Trp Thr Pro Ser Thr Ala Gly Gly Asn Ala Glu Ser Ser Arg 595600 605 Tyr Pro Leu Asn Pro Gln Phe Arg Leu Glu Ile Glu Asn Asp Thr Asp610 615 620 Val Ser Leu Leu Leu Glu Cys Pro Asn Thr Glu Leu Ala Thr HisVal 625 630 635 640 Lys Leu Phe Trp Ser Asn Gly Asn Arg Val Ser Arg ValArg Ser Arg 645 650 655 Asp Ile Ile Ala Asp Ser Gly Asp Tyr Arg Arg GlyGly Ser Leu Val 660 665 670 Glu Lys Lys Ala Leu Glu Pro Gly Ser Tyr ThrIle Val Cys Ser Thr 675 680 685 Phe Ala Pro Asp Gln Leu Gly Arg Phe ThrLeu Trp Val Ser Ser Leu 690 695 700 Val Pro Cys Lys Thr Ser Pro Leu ProPro Glu Ala Ala Gly Arg Arg 705 710 715 720 Thr Val Ile Ser Asp Ile GlyVal Leu Pro Pro Gly Arg Asp Arg Met 725 730 735 Leu Ala Ser Leu Gln ValPro Arg Leu Thr Arg Ile Lys Leu Ile Thr 740 745 750 Arg Ser Arg Gln SerIle Ile Gly Ser His Pro Val Gly Pro Ser Pro 755 760 765 Val Leu Met ThrVal Glu Leu Gly Gln Gly Pro Tyr Lys Gln Ile Leu 770 775 780 Ala Thr SerGlu Asp Gly Thr His Ser Asp Ala Val Ser Gly Val Arg 785 790 795 800 ValGlu Asp Phe Asp Leu Gln Pro Gly Leu Glu Glu Ser Gly Gly Ile 805 810 815Trp Ile Val Ile Glu Arg Ile Gly Gly Pro Gly Gly Gln Val Glu Asp 820 825830 His Phe Glu Val Glu Ala Leu Ala Glu Glu Arg Val Glu Ile Gly Glu 835840 845 Trp Ile Leu Glu Asp Ala 850 (2) INFORMATION FOR SEQ ID NO: 18:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 842 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Met Ser Arg Thr SerSer Ala Pro Ser Gln Lys Ser Leu Ile Ser Arg 1 5 10 15 Ala Leu Lys AlaGlu Arg Asp Val Ile Thr Ala Ser Ser Gln Ser Gln 20 25 30 Ala Leu Asp AlaAla Ile Asp Ala Ala Glu His Tyr Met Lys Ala Leu 35 40 45 Ala Leu Thr SerSer Ser Lys Asp Arg Asn Val Leu Asp Ala Lys Cys 50 55 60 Lys Glu Trp LeuThr Arg Ala Glu Lys Ile Lys Gly Ser Glu Asp Trp 65 70 75 80 Arg Ser ValAla Gln Ser Arg Arg Ser Arg Leu Arg Thr Pro Ala Ser 85 90 95 Thr Arg LysLeu Thr Thr Arg Glu Asp Ile Ile Leu Leu Gln Gly Ala 100 105 110 Lys LeuAsn Gly Phe Ile Phe Pro Pro Trp Lys Ala Glu Pro Ser Leu 115 120 125 ThrGlu Phe Glu Thr Gly Thr Asn Gly Asp Val Leu Phe Thr Asp Lys 130 135 140Pro Asp Leu His Leu Ser Asn Leu Gln Arg Asp Ile Phe Ala Gly Trp 145 150155 160 Lys Arg Pro His Glu Leu Leu Ser Gly Gln Val Asp Asp Ala Gly Met165 170 175 Pro Leu Asn Pro Val Met Thr Val Ser Gly Asn Thr Asp Leu ValGln 180 185 190 Asp Val Leu Thr Asp Cys Ser Val Val Ala Ser Leu Cys AlaThr Thr 195 200 205 Ser Arg Ser Glu Arg Gly Leu Asp Asp Thr Leu Leu ProIle Val Tyr 210 215 220 Pro Cys Ile His Asn Ser Met Lys Ser Asp Ile SerPro Ser Gly Lys 225 230 235 240 Tyr Ile Phe Arg Phe Tyr Phe Asn Gly CysPhe Arg Lys Val Val Ile 245 250 255 Asp Asp Arg Leu Pro Ser Ser Lys ThrSer Arg Ser Leu Tyr Met Ile 260 265 270 Asp Arg Asn His Arg Asn Phe MetTrp Pro Ala Leu Val Glu Lys Ala 275 280 285 Tyr Leu Lys Leu Arg Gly GlyTyr Glu Phe Pro Gly Ser Asn Ser Gly 290 295 300 Thr Asp Leu Trp Val LeuThr Gly Trp Ile Pro Glu Gln Val Phe Leu 305 310 315 320 His Ser Asp GluVal Thr Ala Asp Gln Ile Trp Ser Asp Leu Phe Lys 325 330 335 Ser Phe HisSer Gly Asp Val Leu Leu Thr Ile Gly Thr Gly Lys Leu 340 345 350 Thr GluArg Glu Gln Lys Glu Leu Gly Leu Val Ser Glu His Asp Tyr 355 360 365 AlaIle Leu Asp Met Lys Glu Leu Lys Gly Arg Arg Gln Phe Leu Ile 370 375 380Lys Asn Pro Trp Ala Gly Thr Asp Ala Val Tyr Pro Ala Leu Phe Ala 385 390395 400 Asp Pro Gly Pro Phe Pro Asn Ser Pro Phe Leu Ser Pro Gly Thr Phe405 410 415 Trp Met Asp Cys Glu Met Val Leu Gln Asn Phe Glu Asn Leu TyrLeu 420 425 430 Asn Trp Asn Pro Gly Ile Phe Ala Tyr Gln Glu Asp Ile HisPhe Thr 435 440 445 Trp Asp Leu Ser Thr Gly Lys Gly Met Ala Gly Cys PheVal Lys Asn 450 455 460 Pro Gln Phe Ser Val Tyr Thr Glu Arg Gly Gly ValVal Trp Leu Leu 465 470 475 480 Leu Gly Arg His Leu Arg Thr Ile Glu SerArg Ala Ser Glu Glu Asp 485 490 495 Glu Arg Phe Gly Phe Ile Ser Ile TyrVal Phe Lys Gly Gly Lys Arg 500 505 510 Val Ala Leu Ser Asp Gly Ala LeuHis Arg Gly Pro Tyr Val Asp Ser 515 520 525 Pro Asn Thr Leu Met Lys LeuAsp Val Pro Pro Arg Ser Thr Tyr Thr 530 535 540 Ala Val Val Ser Glu GluSer Leu Pro Arg Val Ser Gln Asn Phe Thr 545 550 555 560 Ile Ser Ala PheSer Asp Ser Pro Val Arg Ile Ser His Ala Pro Asn 565 570 575 Lys Tyr IleCys Val Thr Lys Val Gln Gly Ser Trp Thr Pro Thr Thr 580 585 590 Ala GlyGly Asn Ala Glu Ser Ala Arg Tyr Ser Leu Asn Pro Gln Phe 595 600 605 SerIle Val Leu Ser Asp Pro Thr Asp Ile Ser Ile Val Leu Glu Pro 610 615 620Ser Asp Gln Glu Leu Ala Thr His Val Lys Leu Phe Trp Ser Gly Gly 625 630635 640 Lys Arg Ile Ala Arg Val Arg Ser Arg Asp Ile Val Ala Asp Ser Gly645 650 655 Asp Tyr Arg Arg Gly Gly Ser Leu Val Glu Lys Gln Asp Leu AspPro 660 665 670 Gly Glu Tyr Thr Ile Val Val Ser Thr Phe Ala Pro Asp GlnTyr Gly 675 680 685 Ser Phe Thr Leu Trp Val Ser Thr Asn Ile Thr Cys GluVal Thr Gln 690 695 700 Leu Pro Ser Glu Ala Ala Gly Arg Arg Ala Val LeuSer Asp Ile Gly 705 710 715 720 Val Leu Leu Pro Gly Gln Asp Arg Met LeuAla Pro Leu Thr Thr Pro 725 730 735 Arg Leu Thr Arg Val Lys Leu Ile AlaArg Ser Arg Glu Ser Arg Ile 740 745 750 Gly Asn Arg Pro Val Gly Pro SerPro Leu Leu Met Thr Val Glu Leu 755 760 765 Gly Gln Gly Pro Tyr Lys GluIle Leu Ala Thr Ser Glu Asp Gly Asp 770 775 780 His Ser Asp Ser Ile SerGly Val Arg Val Glu Asp Phe Asp Leu Gln 785 790 795 800 Pro Gly Leu GluGlu Arg Gly Gly Val Trp Ile Val Leu Glu Arg Ile 805 810 815 Gly Gly LeuAla Val Lys Trp Lys Ile Ile Ser Lys Trp Lys Leu Trp 820 825 830 Glu LysArg Glu Trp Arg Leu Gly Asn Gly 835 840 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Gly Ile Leu Leu AlaGly Ser Ala Asn Ser Lys Tyr Ala Phe Leu Gly 1 5 10 15 Ser Leu Arg SerThr Ala Gln Leu Ile Ser Tyr Glu Leu Ile Leu Ser 20 25 30 Ser Val Ile LeuLeu Val 35 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 371 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 20: Met Phe Tyr Ser Leu Thr Ile Ile SerIle Leu Glu Val Leu Leu Val 1 5 10 15 Leu Val Pro Ser Leu Leu Ala ValAla Tyr Val Thr Val Ala Glu Arg 20 25 30 Lys Thr Met Ala Ser Met Gln ArgArg Leu Gly Pro Asn Ala Val Gly 35 40 45 Tyr Leu Gly Leu Leu Gln Ala PheAla Asp Ala Leu Lys Leu Leu Leu 50 55 60 Lys Glu Tyr Val Ala Leu Thr GlnAla Asn Met Thr Leu Phe Phe Leu 65 70 75 80 Gly Pro Val Ile Thr Leu IlePhe Ser Leu Leu Gly Tyr Ala Val Ile 85 90 95 Pro Tyr Gly Pro Ser Leu ValIle Gln Asp Val Asn Leu Gly Ile Leu 100 105 110 Tyr Met Leu Ala Val SerSer Leu Ala Thr Tyr Gly Ile Leu Leu Ala 115 120 125 Gly Trp Ser Ala AsnSer Lys Tyr Ala Phe Leu Gly Ser Leu Arg Ser 130 135 140 Ala Ala Gln LeuIle Ser Tyr Glu Leu Val Leu Ser Ser Ala Ile Leu 145 150 155 160 Leu ValIle Met Leu Thr Gly Ser Phe Asn Leu Gly Val Asn Thr Glu 165 170 175 SerGln Arg Ala Val Leu Phe Val Leu Pro Leu Leu Pro Ile Phe Ile 180 185 190Ile Phe Phe Ile Gly Ser Ile Ala Glu Thr Asn Arg Ala Pro Phe Asp 195 200205 Leu Ala Glu Ala Glu Ser Glu Leu Val Ser Gly Phe Met Thr Glu His 210215 220 Ala Ala Val Val Phe Val Phe Phe Phe Leu Ala Glu Tyr Gly Ser Ile225 230 235 240 Val Leu Met Cys Ile Leu Thr Ser Ile Leu Phe Leu Gly GlyTyr Leu 245 250 255 Phe Ile Asn Leu Lys Asp Val Phe Asn Ile Leu Asp PheVal Tyr Ser 260 265 270 Asn Leu Phe Ile Phe Glu Ile Asn Trp Met Val SerGlu Arg Ser Tyr 275 280 285 Thr Glu Asp Phe Phe Asn Asn Tyr Lys Ser IleLeu Glu Gly Trp Leu 290 295 300 Tyr Gly Trp Ile Ile Gly Leu Lys Ser SerIle Met Ile Phe Ile Phe 305 310 315 320 Ile Leu Gly Arg Ala Ser Phe ProArg Ile Arg Tyr Asp Gln Leu Met 325 330 335 Gly Phe Cys Trp Thr Val LeuLeu Pro Ile Ile Phe Ala Leu Ile Ile 340 345 350 Leu Val Pro Cys Ile LeuGlu Ser Phe Tyr Ile Leu Pro Trp Asn Leu 355 360 365 Asn Leu Phe 370 (2)INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GGTTGCATGCTCTAGACTTC GTCACCTTAT TAGCCC 36 (2) INFORMATION FOR SEQ ID NO: 22: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO : 22: TTCGCGCGCA TCAGTCTCGA GATCGTGTGT CGCGAGTACG40 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:GATCTCGAGA CTAGTGCGCG CGAACAGACA TCACAGGAAC C 41 (2) INFORMATION FOR SEQID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 24: CAACATATGC GGCCGCGAAT TCACTTCATTCCCACTGCGT GG 42 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 6800 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 25: TATTCTGCTA GTAGTTAGAT CTACTGAGGGGGTATAACTC TTCGGTAGCC GGTGCGTATG 60 CAGTTGTATT GTGACTGATA GATGAGAAGAAGGGCGTTTA CTGGTTGCAA GAGAATGATG 120 TTATGCTTTG CATATAAGAG ATGGAATTATTACTGATATG TGTCTAAAAT TACAATGATT 180 TGTTTTACTG AAACTGAGTA TAATGCCAGTTTGGATGTAG CCGCGTAGGT GATACTTAAT 240 CCGGACCAAA TTATGAGACC TCGATAGTACAATGCTATTG GACTTGTAAG ATAATAGTCT 300 AGTTCTTCAT AGACAAACCT GAAAAAGGAAGGACTCGAAG TCATTTCGAC AAGCCATAGT 360 ATGATTAGGC ATCTAGGAGC TGAACGGAAACGTCGTATGC GAGAGGGCCA GAACCAAAAT 420 ACGACAGTAG GAGGATTCCT ACCCCTTGGTATATATGGAA ACCGGTTTTA AAGACTGGGC 480 GTCCTCGTAC CTCACAGTCG AGCATGCCAGACGTTGGCGC ACACATTCCC CAGATCGGCT 540 GGAGCCTATC GCAATGCAAC CGTACCCACCCGGCGTNTAC TATATCTGAG CATTCCTGCT 600 GCAAGTCGTG GAGGTTGTTC CAGCCAATGAGGTCCGTTGA GCTTCTTTTT CCGATATGGT 660 GATAGCTGAC AGTGTACTAG TGTCGACTGCACAGGTGCCT CGCGGAAATG TGAGATCGGA 720 GCATGTCTTG CTTCAGCGAC CGAAGTATGGACGAATGTCC GCCATCTTGA AGAATGGTGA 780 CTACCGGAGG ATTGTCTCCA GCTCAGGACTATGTATATGC CACGATGCCA CGGCTTTGTG 840 TGATAGAGTT CAAGAGCCGA GATTTACAACGGTGATTACT ATCGCAGATA TCAGCACAGT 900 ACCGGCAAGA TGGTCAACTG GAGCAACATACTACCCACAT TAACCGCCTT CATCGCTTTT 960 ATCCTGGGCA TGCTATGCCT CTTTGCAGGAACGAAAACAA ACCTTTTATT AGATACAGAT 1020 GTCTTCACGG TGTGTGAGCC TCCGTCACACTTGCTTGTAC GAGCGGGGAC TAACGACAGG 1080 CGAAGATATA TACGACAAGT ATAAGCAATGGCACGGGGAT GCGGGACTTC TACTCGATAT 1140 ATGTCATGTC TTACTGTGAG GGATTCCTGCATGCAGAAAA TCGAAACCTA ACCGGATGCT 1200 CACACCCGTC ACTACTGTTC TCCTTCAATGCGACAGAAGC GTTAACGAAA GATGCTGGCA 1260 ACAACACCTC GTTATCCAGC CTGGGATGGCCGAGTTCCAT CACCGATGAT CTACGCACGT 1320 TCGGTGCTAC CAGCCAGAGT ATGGGTGTCTTCTACTGTAT TGGGATAGGA TTAGCGGGAC 1380 TGGCGGTTTT GGAACGATTG TGGTTCGTGATCGCGAAAGG GCCGAGACAG ACGGTTGTAG 1440 AAGTTTCTTC TCTTATGGTA GGCTCCATTGAACGTTCTAG ATCCCGTCCG GTACTAAGAA 1500 GGCCCACAGC TCAGTTTCAC TATGCTCAGCATACCGTCTA TCATCGCAAC GGTCGTTGCC 1560 TTACAATTTG TGAGCCTCAT CAATCGTCACGGAGAGGAGT CTGGTGTGAC AGCGAGATAT 1620 GGACATCAAT TTTTAGGAAT GACGTGGGCAGCCGTCGGGT TGTTGCTGGT CGGAAGCACT 1680 GTCAGTTTAC TGACGGTATT GGTGGACCGCAACCGATCGG CAGACCAGTA TGAACCGGTG 1740 GCAGAACCGA AGACGGTGGC CGAGGATTCGGACTCGGTAG CGTCGAACCA GAAGGGGGAC 1800 TAAGAAAGGA ACAGAATGGA GAGGAATAATCATAAGAGAA AAAAAGGGGG AAATTAACCA 1860 AAGCAGGAAA AGTAGGGAAA AAAAAAAGAAGAAGACCGGA GAAAGCCAAG GAAAGGGAAC 1920 GAATCGGGAG GAGTGTTTCT TGTTTGCAAATACGTTGGAT TGGAGCCCAA TATTGAATAT 1980 ACTCCGTACT GTAGTCGAAA GAAAGGACAAGAGCCCCACC AAACCTCGAC CGTTCCATAG 2040 CAGAAATTCC ATGACTATCT GTTAATATTTTCTGATCGAT GATGTTGTAA CAGTGGTTGA 2100 AGTGGACCTT ATTTTTCGCA ATACAATAGCAGCATGGCTT GTGGGAAGGG GTCTTGAGAT 2160 CATAGTCATA GTGTAAGAAA AAATTGGGTCTCCCGCGGAT GGCGACGTCA CGAGTGGACC 2220 CGGGAAAAGG TCTCGCCAAA TGAGGCAACCCGGTTCCCTC CGTTCCAACC GCTATGCCTG 2280 CGATTCTATC AATGATAGTG GGTCACTCGATGGATTCAAG GGATGTCAAA TGATGACGTT 2340 AAGTTGACTA CGACTGTTGC TGTCATGCACTGGAAATTTA TCGGGAGGAA TTGACCAGTC 2400 TTATTCGCGG AGGAGGGAGT GAAGAGACTAGGAGTCGTTC AGTTCAGTCT TGCAGGAAAT 2460 CCACTCGGGG ATGAGCAGGG GTTAACCTCATGGACGACTG AACTACTCCG TACAGGACGG 2520 AGTACAGAAT GTCGAATTCA CCCGTTCGGCTAAAGATGAG TCCGTTTTAT GGTCACCTAG 2580 CGGAGTGCAA CCCCGACTGG ATAACTGTTAGGAAAAGAGA GAATTAGGAT CGGGTGACAA 2640 TTGGGTCTCT AAGTTCCTGC GTAACGTGATACCGAATCAA ATCCATGACC CTATCGCCAG 2700 GACGATCACG GAATGGTCCG GATCAGAAATTCTGAGGTTC TGGTTGGATT GATGACCTGA 2760 ACTAATACCC AATATCATGA CGAAAACCAATCCCCTCATT TCCTGTTTTT GCACGGGAAT 2820 AGCCACAATT TCCCCCCCCC CCCCCCCCCCCCAGAAAACC GAAATGAAGT CTGAGCCCTC 2880 CGGAAGACTG CGTTCCTAGC CCCCATGTGTTTAGTTGATA CTATTCCCAT AGGCACGTTT 2940 CCCCCCTTCC CCACTTGAGT TCCACCAGTCGTGAATGAGG AGGTTCCATT CTCGCCCAGA 3000 GTTTGGTTTC TTTGCCGTTC CATACAAGGCGTCACTGCAT CATTCCTTCC CTTTTCATTC 3060 ACCCCTCTCT TGTCCACCAT CGTGAAATGTTTCTGTAGTA CATTTAATAA ATACCCCTCG 3120 TTACCCCTCT TTCTGTTTCC CAAGAAATCAACATCATCAT CAACAACAAC AACAACAATC 3180 ACCCTCCCAC TTCACAGGTT CTCTTTCTGATACCCATCCT TCTGTCTCTC ATCTACTACC 3240 ACTACTTTCA TATACTCTCT TCTATCCTACTTCATCACCA TCACAACCTT CTTCCCCATT 3300 CTTGTTTCAA CCCAACATCA ATATATTACCGTTGTCAACC ATCCATCATG GGTAAAAAGG 3360 CTATCCAGTT TGGCGGTGGA AACATTGGCCGTGGCTTTGT GGCTGAGTTT CTCCACGCTG 3420 CCGGCTATGA AGTCGTCTTC ATTGATGTCATGGATAGCGT CATCAACTCT TTGCAACAGA 3480 CCCCGTCGTA CGACGTCACG GAGGTCAGCGAAGAGGGTGA AAGCACCAAG ACCATCACCA 3540 ACTATCGCGC CATCAACTCC AAGACGCATGAGGCCGACGT CGTTCAGGAG ATCGCATCGG 3600 CAGATGTGGT TACCTGTGCT GTCGGTCCCAACATCCTTAA GTTCATCGCG CCAGTCATTG 3660 CCAAAGGTAT TGATGCGCGC ACCGAAGAGAGACCCGTGGC TGTGATCGCC TGTGAGAACG 3720 CTATCGGCGC TACAGATACC TTGCACGGCTACATCAAGCA GCACACCAAC CCTGACCGTC 3780 TGGAGACCCT CTCTGAGCGT GCCCGTTTTGCCAACTCGGC TATCGACCGC ATCGTCCCCA 3840 ACCAGCCCCC GAACAGTGGT CTCAATGTTCGCATCGAGAA GTTCTACGAG TGGGCCGTGG 3900 AGAAGACTCC ATTTGGCGAA TGGGGTCACCCCGACATCCC TGCCATCCAC TGGGTGGACC 3960 ACCTCGAACC TTACATCGAA CGCAAGCTCTTCACCGTCAA CACTGGCCAT GCTACCACCG 4020 CCTACTATGC TCACAAGCGT GGCAAGAAGATGATCGCCGA GGCCCTCGAA GACCCAGAGA 4080 TCCGCGAGAC TGTGCACAAG GTGCTCGAGGAGACTGCTTC CCTCATTGTA TCCAAGCATG 4140 AGATCTCGGA GCAGGAGCAG AAGGAATACGTTGACAAGAT TGTCAGCCGT ATCTCCAACC 4200 CCTATCTCGA GGACAACGTT GAGCGTGTGGGACGTGCTCC TCTCCGCAAA CTGTCTCGCA 4260 AGGAACGGTT CATTGGACCT GCTTCGCAGCTCGCAGAGCG CGGCCAGAAG TTCGATGCTC 4320 TCCTGGGCGC CATCGAGATG GCTCTTCGCTTCCAGAACGT CCCAGGCGAC GAGGAGAGTT 4380 CCGAGCTTGC TCGCATTTTG AAGGAGAACTCGGCCGAGGA TGCCACCTCG CAGCTCACCG 4440 GATTGGAGAA AGACCACCCA CTCTACTCTCATGTGGTTGA GCGTGTGTCC ACGGTCCAGC 4500 AAGGCTCCAA ATCAGTGCTG TGATTCTCGATCGTTTTCCA CACCACCACA CTCCTTTTTA 4560 TCACCAGAAA ACGAAGGGTT CCGAGTCCATCACCAATATG GATCGCCCGA GGGATATTGG 4620 ATCTGATATC AAACTGTTCT GTCCGCTGGCCGGGCATGAA CTGCATGGGA TACGGCGAAC 4680 ATATGAAATA ACCCCCAATT CCCATAAGTATTACATATTA TGGAACCACA GCCGGTGTCT 4740 GTAAATGTCG GTTCAACTCG AAGATGGCCGATGCAATCGG CCCGTAGGGT ATATGGTCTG 4800 GCGCCACCTC GGCCGCCGGC TTCCCCCTTTTTATAGATGT GGCGAATAAA ACACCGGATG 4860 TTTTGTGTGT CAGGGGAATG GTGGCAGTGGTGTTATGAGT CATTGTGAAG TGAGTAGTGA 4920 GTAGATTTGG TGGGGATTTT CATAGATGGTGGTTTGAAGG TCTTGGGTTT CTGGGGTTTA 4980 TCCGCGTATA TTCTGCTAGT AGTTAGATCTACTGAGGGGG TATAACTCTT CGGTAGCCGG 5040 TGCGTATGCA GTTGTATTGT GACTGATAGATGAGAAGAAG GGCGTTTACT GGTTGCAAGA 5100 GAATGATGTT ATGCTTTGCA TATAAGAGATGGAATTATTA CTGATATGTG TCTAAAATTA 5160 CAATGATTTG TTTTACTGAA ACTGAGTATAATGCCAGTTT GGATGTAGCC GCGTAGGTGA 5220 TACTTAATCC GGACCAAATT ATGAGACCTCGATAGTACAA TGCTATTGGA CTTGTAAGAT 5280 AATAGTCTAG TTCTTCATAG ACAAACCTGAAAAAGGAAGG ACTCGAAGTC ATTTCGACAA 5340 GCCATAGTAT GATTAGGCAT CTAGGAGCTGAACGGAAACG TCGTATGCGA GAGGGCCAGA 5400 ACCAAAATAC GACAGTAGGA GGATTCCTACCCCTTGGTAT ATATGGAAAC CGGTTTTAAA 5460 GACTGGGCGT CCTCGTACCT CACAGTCGAGCATGCCAGAC GTTGGCGCAC ACATTCCCCA 5520 GATCGGCTGG AGCCTATCGC AATGCAACCGTACCCACCCG GCGTCTACTA TATCTGAGCA 5580 TTCCTGCTGC AAGTCGTGGA GGTTGTTCCAGCCAATGAGG TCCGTTGAGC TTCTTTTTCC 5640 GATATGGTGA TAGCTGACAG TGTACTAGTGTCGACTGCAC AGGTGCCTCG CGGAAATGTG 5700 AGATCGGAGC ATGTCTTGCT TCAGCGACCGAAGTATGGAC GAATGTCCGC CATCTTGAAG 5760 AATGGTGACT ACCGGAGGAT TGTCTCCAGCTCAGGACTAT GTATATGCCA CGATGCCACG 5820 GCTTTGTGTG ATAGAGTTCA AGAGCCGAGATTTACAACGG TGATTACTAT CGCAGATATC 5880 AGCACAGTAC CGGCAAGATG GTCAACTGGAGCAACATACT ACCCACATTA ACCGCCTTCA 5940 TCGCTTTTAT CCTGGGCATG CTATGCCTCTTTGCAGGAAC GAAAACAAAC CTTTTATTAG 6000 ATACAGATGT CTTCACGGTG TGTGAGCCTCCGTCACACTT GCTTGTACGA GCGGGGACTA 6060 ACGACAGGCG AAGATATATA CGACAAGTATAAGCAATGGC ACGGGGATGC GGGACTTCTA 6120 CTCGATATAT GTCATGTCTT ACTGTGAGGGATTCCTGCAT GCAGAAAATC GAAACCTAAC 6180 CGGATGCTCA CACCCGTCAC TACTGTTCTCCTTCAATGCG ACAGAAGCGT TAACGAAAGA 6240 TGCTGGCAAC AACACCTCGT TATCCAGCCTGGGATGGCCG AGTTCCATCA CCGATGATCT 6300 ACGCACGTTC GGTGCTACCA GCCAGAGTATGGGTGTCTTC TACTGTATTG GGATAGGATT 6360 AGCGGGACTG GCGGTTTTGG AACGATTGTGGTTCGTGATC GCGAAAGGGC CGAGACAGAC 6420 GGTTGTAGAA GTTTCTTCTC TTATGGTAGGCTCCATTGAA CGTTCTAGAT CCCGTCCGGT 6480 ACTAAGAAGG CCCACAGCTC AGTTTCACTATGCTCAGCAT ACCGTCTATC ATCGCAACGG 6540 TCGTTGCCTT ACAATTTGTG AGCCTCATCAATCGTCACGG AGAGGAGTCT GGTGTGACAG 6600 CGAGATATGG ACATCAATTT TTAGGAATGACGTGGGCAGC CGTCGGGTTG TTGCTGGTCG 6660 GAAGCACTGT CAGTTTACTG ACGGTATTGGTGGACCGCAA CCGATCGGCA GACCAGTATG 6720 AACCGGTGGC AGAACCGAAG ACGGTGGCCGAGGATTCGGA CTCGGTAGCG TCGAACCAGA 6780 AGGGGGACTA AGAAAGGAAC 6800 (2)INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:391 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 26: Met Gly Lys Lys Ala Ile Gln Phe Gly Gly Gly Asn Ile Gly ArgGly 1 5 10 15 Phe Val Ala Glu Phe Leu His Ala Ala Gly Tyr Glu Val ValPhe Ile 20 25 30 Asp Val Met Asp Ser Val Ile Asn Ser Leu Gln Gln Thr ProSer Tyr 35 40 45 Asp Val Thr Glu Val Ser Glu Glu Gly Glu Ser Thr Lys ThrIle Thr 50 55 60 Asn Tyr Arg Ala Ile Asn Ser Lys Thr His Glu Ala Asp ValVal Gln 65 70 75 80 Glu Ile Ala Ser Ala Asp Val Val Thr Cys Ala Val GlyPro Asn Ile 85 90 95 Leu Lys Phe Ile Ala Pro Val Ile Ala Lys Gly Ile AspAla Arg Thr 100 105 110 Glu Glu Arg Pro Val Ala Val Ile Ala Cys Glu AsnAla Ile Gly Ala 115 120 125 Thr Asp Thr Leu His Gly Tyr Ile Lys Gln HisThr Asn Pro Asp Arg 130 135 140 Leu Glu Thr Leu Ser Glu Arg Ala Arg PheAla Asn Ser Ala Ile Asp 145 150 155 160 Arg Ile Val Pro Asn Gln Pro ProAsn Ser Gly Leu Asn Val Arg Ile 165 170 175 Glu Lys Phe Tyr Glu Trp AlaVal Glu Lys Thr Pro Phe Gly Glu Trp 180 185 190 Gly His Pro Asp Ile ProAla Ile His Trp Val Asp His Leu Glu Pro 195 200 205 Tyr Ile Glu Arg LysLeu Phe Thr Val Asn Thr Gly His Ala Thr Thr 210 215 220 Ala Tyr Tyr AlaHis Lys Arg Gly Lys Lys Met Ile Ala Glu Ala Leu 225 230 235 240 Glu AspPro Glu Ile Arg Glu Thr Val His Lys Val Leu Glu Glu Thr 245 250 255 AlaSer Leu Ile Val Ser Lys His Glu Ile Ser Glu Gln Glu Gln Lys 260 265 270Glu Tyr Val Asp Lys Ile Val Ser Arg Ile Ser Asn Pro Tyr Leu Glu 275 280285 Asp Asn Val Glu Arg Val Gly Arg Ala Pro Leu Arg Lys Leu Ser Arg 290295 300 Lys Glu Arg Phe Ile Gly Pro Ala Ser Gln Leu Ala Glu Arg Gly Gln305 310 315 320 Lys Phe Asp Ala Leu Leu Gly Ala Ile Glu Met Ala Leu ArgPhe Gln 325 330 335 Asn Val Pro Gly Asp Glu Glu Ser Ser Glu Leu Ala ArgIle Leu Lys 340 345 350 Glu Asn Ser Ala Glu Asp Ala Thr Ser Gln Leu ThrGly Leu Glu Lys 355 360 365 Asp His Pro Leu Tyr Ser His Val Val Glu ArgVal Ser Thr Val Gln 370 375 380 Gln Gly Ser Lys Ser Val Leu 385 390 (2)INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:195 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 27: Met Val Asp Gln Ala Gln Asp Thr Leu Arg Pro Asn Asn Arg LeuSer 1 5 10 15 Asp Met Gln Ala Thr Met Glu Gln Thr Gln Ala Phe Glu AsnArg Val 20 25 30 Leu Glu Arg Leu Asn Ala Gly Lys Thr Val Arg Ser Phe LeuIle Thr 35 40 45 Ala Val Glu Leu Leu Thr Glu Ala Val Asn Leu Leu Val LeuGln Val 50 55 60 Phe Arg Lys Asp Asp Tyr Ala Val Lys Tyr Ala Val Glu ProLeu Leu 65 70 75 80 Asp Gly Asp Gly Pro Leu Gly Asp Leu Ser Val Arg LeuLys Leu Ile 85 90 95 Tyr Gly Leu Gly Val Ile Asn Arg Gln Glu Tyr Glu AspAla Glu Leu 100 105 110 Leu Met Ala Leu Arg Glu Glu Leu Asn His Asp GlyAsn Glu Tyr Ala 115 120 125 Phe Thr Asp Asp Glu Ile Leu Gly Pro Phe GlyGlu Leu His Cys Val 130 135 140 Ala Ala Leu Pro Pro Pro Pro Gln Phe GluPro Ala Asp Ser Ser Leu 145 150 155 160 Tyr Ala Met Gln Ile Gln Arg TyrGln Gln Ala Val Arg Ser Thr Met 165 170 175 Val Leu Ser Leu Thr Glu LeuIle Ser Lys Ile Ser Leu Lys Lys Ala 180 185 190 Phe Gln Lys 195 (2)INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:366 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 28: Met Ile Ala Leu His Phe Gly Ala Gly Asn Ile Gly Arg Gly PheIle 1 5 10 15 Gly Ala Leu Leu His His Ser Gly Tyr Asp Val Val Phe AlaAsp Val 20 25 30 Asn Glu Thr Met Val Ser Leu Leu Asn Glu Lys Lys Glu TyrThr Val 35 40 45 Glu Leu Ala Glu Glu Gly Arg Ser Ser Glu Ile Ile Gly ProVal Ser 50 55 60 Ala Ile Asn Ser Gly Ser Gln Thr Glu Glu Leu Tyr Arg LeuMet Asn 65 70 75 80 Glu Ala Ala Leu Ile Thr Thr Ala Val Gly Pro Asn ValLeu Lys Leu 85 90 95 Ile Ala Pro Ser Ile Ala Glu Gly Leu Arg Arg Arg AsnThr Ala Asn 100 105 110 Thr Leu Asn Ile Ile Ala Cys Glu Asn Met Ile GlyGly Ser Ser Phe 115 120 125 Leu Lys Lys Glu Ile Tyr Ser His Leu Thr GluAla Glu Gln Lys Ser 130 135 140 Val Ser Glu Thr Leu Gly Phe Pro Asn SerAla Val Asp Arg Ile Val 145 150 155 160 Pro Ile Gln His His Glu Asp ProLeu Lys Val Ser Val Glu Pro Phe 165 170 175 Phe Glu Trp Val Ile Asp GluSer Gly Phe Lys Gly Lys Thr Pro Val 180 185 190 Ile Asn Gly Ala Leu PheVal Asp Asp Leu Thr Pro Tyr Ile Glu Arg 195 200 205 Lys Leu Phe Thr ValAsn Thr Gly His Ala Val Thr Ala Tyr Val Gly 210 215 220 Tyr Gln Arg GlyLeu Lys Thr Val Lys Glu Ala Ile Asp His Pro Glu 225 230 235 240 Ile ArgArg Val Val His Ser Ala Leu Leu Glu Thr Gly Asp Tyr Leu 245 250 255 ValLys Ser Tyr Gly Phe Lys Gln Thr Glu His Glu Gln Tyr Ile Lys 260 265 270Asn Gln Arg Ser Leu Leu Lys Ser Phe His Phe Gly Arg Cys Asp Pro 275 280285 Arg Ser Glu Val Thr Ser Gln Lys Thr Gly Arg Lys Cys Arg Leu Val 290295 300 Gly Pro Ala Lys Lys Ile Lys Glu Pro Asn Ala Leu Ala Glu Gly Ile305 310 315 320 Ala Ala Ala Leu Arg Phe Asp Phe Thr Gly Asp Pro Glu AlaVal Glu 325 330 335 Leu Gln Ala Leu Ile Glu Glu Lys Asp Thr Ala Ala TyrPhe Lys Arg 340 345 350 Cys Ala Ala Phe Ser Pro Met Asn Arg Cys Thr ProSer Phe 355 360 365 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 3300 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 29: GGATTGGCTG ACTGCGTGTT GTTACTCCAG GGATTGCGAC GAGCACTTCCTAAGCCACCG 60 TGCGACGGAG GCGACTCTGA GCGTGACGAT GCCCTAGCGA ACCCGCGGAAGCCCATCGTG 120 GGGCGAAAAT CTTCTTCGTC GGAGCTCATG TCGGCGATTT TACGTTTTTGGCCGGCGGAT 180 GGTGAGGCCG GGGGAGAATC CGAATCCATG ACCGATCGCA GATGTCAGGATAAGGTGTAA 240 GTAGTAACTC AGAGTCGTCG GAGAGGTTCG AAAGGCAATG AAACGGTCAAACGACACGTT 300 TGAGAGCCAC GAAGGAGCTG TGGGTTGAGA TACGCACGAT AACGAGAAAGGAAAGTTGAT 360 TATCGGACAT TTCGGCGCGG GGAAAATTCA AGTCCGAGGG GCCGAGCAACAATGACGTTC 420 GTTGCATCGA ATCTCCCTTC CGGTTATTTT TCCCTCTTCT TCTCCTCTTCTTCTTTTCTT 480 CTTTACCCTC TCCTCTCTTT GGCATTTCGT CACTACTTTG TAACGTAACTCAATTCTATT 540 GATACATAAA AATCACATAT CAACTATGGC TGCCTCTCTT ATCCGTACCTCTGCCCGTAC 600 CGCTCTTCGC GCTGGAGCTT CGGCTACTCC TAAAGCTGCG GGTGTTGCGGGTTTGACCTT 660 TGCCCGTGGC AAGGCCACTC TGCCTGACCT GGCTTGTATG GCTCTCCCCTTCCCTTGATG 720 TCGTCAATTT GCCCCTCTGT TGTGTTATCT TCCGTTTTGT CATCTTTCTCGGCTATTTTG 780 GCAGTGCGAA TGAGTAGATG GGTTACGCTT GTCGCTCATG ACGCCCCGGAAGCACGTAAT 840 GCAATGGTTG GTTGACTGAA TAACAGATGA CTATGGCGCC CTTGAGCCCTCTATCTCCGG 900 AAAGATCATG GAGCTTCACC ACAAGAACCA CCACCAGACC TATGTCAACAGCTACAACAC 960 CGCCATCGAA CAGCTCCAGG AGGCCGTCGC CAAGGAGGAC ATCACCACTCAGATCAACCT 1020 CAAGCCCCTG ATCAACTTCC ACGGTGGTGG CCACATCAAC CACACTCTTTTCTGGGAGAA 1080 CCTTGCCCCT AAGAGCCAGG GCGGTGGTGA GCCCCCATCT GGAGCTTTGGCCAAGGCCAT 1140 CGACGAAAGC TTCGGCAGCT TGGGAGAGTT CCAGAGCAAG ATGAACGCCGCCCTCGCTGG 1200 TATTCAGGGA AGCGGATGGG CTTGGCTCGT CAAGGACAAG CAGACCGGAAACATCGGCAT 1260 CAAGACCTAT GCCGTAAGTT CCTCCTTGTG AGCGCCTAAG GATACAGGTAGCTAACTCCC 1320 GACCAGAACC AGGACCCTGT CGTTGGTCAG TTCCAGCCTC TTCTCGGTATTGATGCTTGG 1380 GAGCACGCCT ACTAGTAAGT TTTCTTGGAC TAGATATCTA CCAAGCAATAACTAATGCCG 1440 TGTTAGCCTT CAATACCAGA ACCGCAAGGC TGAGTACTTC AGCGCCATCTGGGACGTCAT 1500 CAACTGGAAG GCGGTTGAGA AGCGCTTCTC GTAAGCGTGC AAAAGTGTTGTGAATTGACG 1560 CAGCTTGATG AGCGCTTTGT TTCAGTTGTG CCCAGAGTGA TACTGTGTAATGTCTGATCA 1620 AGCTGTACTT GTAGCCCTAA TGCAATTGGA TACGCCTCGT GTATATATAAACTCATGTTC 1680 GTTGAACGTA AATAATTTTG GGGAAGCTGC ACCAGCCACA GTGGCTGGATCACATGCTCC 1740 CGTAGCATTC CCGCAGTTTC CGGCAAGCTT ATTTTCTTAG TTTGGGATCCGCTCCGCCCT 1800 CTCCGGATCT TCTTCCCTCA TCTCACCTCT CAAGCGATCA ATTCTCTCGAAATGTCTGCA 1860 GAAAGCCCGG GAGAAAAGCG CGGTGGGTTT CGGGCGTTCT TCGCCGGCGCCCTCCGACCT 1920 AAGAAATCCC GTCAGGTCCT CCGAAAGGCA TCGACACCGA ATCTAAAGGAAGGTCTACAA 1980 AGCAAAGATG ACGTCCCGGC GATGCCTTCA CTGACCCCAT TGGAGGCCCACCGACTCAAA 2040 TACCGAGAAG TAAATCTTCA GAAAGACACA CAGCTAGGCG AAACCCACGATCATACCGCA 2100 ATGCTGCATT CAATCGGTGT TGGAGAGCTC GATCCGTCCG ATCCACACGCGCAACTACAC 2160 GAATTCGACA ATAGACCCCC AGGCGAGCCT ATGATTGCGA GCTTAACATCGGACCTCTGG 2220 GCCAAGGTCA CCGAGTATCT CAATCCCGCC GAAAGAGCCA GTCTTGCCTTCTCCAGCCGA 2280 ACACTATACG CTCGTCTGGG CCGCGAGCCC TGGATAACAA TAAACCTCCCAGAAAACCAC 2340 GACTACAAAG CGGACTTTCT CATCTCCCAA GATAGACTAC TCCCTCACCATCTCCTCTGT 2400 TTCCCCTGCG GCAAATACCA CCGCCGCACA CAAGAAGGCT ACGAAAAGCTCCAACCCGCA 2460 GACATAATCA ACCCGCTCTT CGATTGCCCC AACGCCCGCA ACAACGCCCTCCCAGCACCC 2520 CGCCACCGCA TCACCCACGG CCGAGTCCTT TACTTCACCT TCCACCAGCTAGTCATGCGC 2580 GCATACCGAT TTGGACCCCG CTACGGCATC TCAGCCGACT CTCTATCCCGTCGCTGGCGC 2640 CGGGACGGCT GGTCCCACCA AACCCGATAC CACATCCATC AGGGTCGACTGCTCATGCGA 2700 GTCGTGAGCA CCTGCTTCGC CGAACCAGGC CTCAGCGCCA GCCAACAGCGACTCCTCCTC 2760 TACTCGCGCG ACGACTACTG GCCGTACTTC TCCGTCTGCG CGCACTGGCGGGATGGCGAA 2820 CTTATGAACG TTTGCAAATG CGCCCTCGGC CACATCCCCG TCCCCCGCACCACGAACGGC 2880 CTGCAGGGCC TCGAACACCG CGCAAAAGAT ATGTACCACC GTCGAGAGCACAATCCCAAC 2940 GCCCTCGCGT CGCTCTGCGG TAAGTGTCGA CCTATGCGTC GCTGCCCCGAGTGTCCCTCC 3000 GAGTATCTGG TCGAGGTCAA GCTCACCGAG GACCGGAGTG GTTCGCATCGCAACTTATTC 3060 CGGCATGCGA TTGTGGTGAC ACGGTGGAGT GATTTGGGGG ATGGGCGGTCGCCGCGGCTA 3120 TCGAAGGAGT GGGCGGCGAT TAATGGGGAC GAGGCGGGTG AGGGGTATGATTCTTTTGAG 3180 AAAATAGGGA AGAGGGCTAT TTCGGGGATT TTTGAGTCGG CTATTACCGATGATACTTTG 3240 CCTGGGCAGA GGATTCTTTC AATGAATCCT AAGGGAAAGA AGTTGGGTGAGGCTGGGAAT 3300 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 230 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 30: Met Ala Ala Ser Leu Ile Arg Thr SerAla Arg Thr Ala Leu Arg Ala 1 5 10 15 Gly Ala Ser Ala Thr Pro Lys AlaAla Gly Val Ala Gly Leu Thr Phe 20 25 30 Ala Arg Gly Lys Ala Thr Leu ProAsp Leu Ala Tyr Asp Tyr Gly Ala 35 40 45 Leu Glu Pro Ser Ile Ser Gly LysIle Met Glu Leu His His Lys Asn 50 55 60 His His Gln Thr Tyr Val Asn SerTyr Asn Thr Ala Ile Glu Gln Leu 65 70 75 80 Gln Glu Ala Val Ala Lys GluAsp Ile Thr Thr Gln Ile Asn Leu Lys 85 90 95 Pro Leu Ile Asn Phe His GlyGly Gly His Ile Asn His Thr Leu Phe 100 105 110 Trp Glu Asn Leu Ala ProLys Ser Gln Gly Gly Gly Glu Pro Pro Ser 115 120 125 Gly Ala Leu Ala LysAla Ile Asp Glu Ser Phe Gly Ser Leu Gly Glu 130 135 140 Phe Gln Ser LysMet Asn Ala Ala Leu Ala Gly Ile Gln Gly Ser Gly 145 150 155 160 Trp AlaTrp Leu Val Lys Asp Lys Gln Thr Gly Asn Ile Gly Ile Lys 165 170 175 ThrTyr Ala Asn Gln Asp Pro Val Val Gly Gln Phe Gln Pro Leu Leu 180 185 190Gly Ile Asp Ala Trp Glu His Ala Tyr Tyr Leu Gln Tyr Gln Asn Arg 195 200205 Lys Ala Glu Tyr Phe Ser Ala Ile Trp Asp Val Ile Asn Trp Lys Ala 210215 220 Val Glu Lys Arg Phe Ser 225 230 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 233 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Met Phe Ala Lys ThrAla Ala Ala Asn Leu Thr Lys Lys Gly Gly Leu 1 5 10 15 Ser Leu Leu SerThr Thr Ala Arg Arg Thr Lys Val Thr Leu Pro Asp 20 25 30 Leu Lys Trp AspPhe Gly Ala Leu Glu Pro Tyr Ile Ser Gly Gln Ile 35 40 45 Asn Glu Leu HisTyr Thr Lys His His Gln Thr Tyr Val Asn Gly Phe 50 55 60 Asn Thr Ala ValAsp Gln Phe Gln Glu Leu Ser Asp Leu Leu Ala Lys 65 70 75 80 Glu Pro SerPro Ala Asn Ala Arg Lys Met Ile Ala Ile Gln Gln Asn 85 90 95 Ile Lys PheHis Gly Gly Gly Phe Thr Asn His Cys Leu Phe Trp Glu 100 105 110 Asn LeuAla Pro Glu Ser Gln Gly Gly Gly Glu Pro Pro Thr Gly Ala 115 120 125 LeuAla Lys Ala Ile Asp Glu Gln Phe Gly Ser Leu Asp Glu Leu Ile 130 135 140Lys Leu Thr Asn Thr Lys Leu Ala Gly Val Gln Gly Ser Gly Trp Ala 145 150155 160 Phe Ile Val Lys Asn Leu Ser Asn Gly Gly Lys Leu Asp Val Val Gln165 170 175 Thr Tyr Asn Gln Asp Thr Val Thr Gly Pro Leu Val Pro Leu ValAla 180 185 190 Ile Asp Ala Trp Glu His Ala Tyr Tyr Leu Gln Tyr Gln AsnLys Lys 195 200 205 Ala Asp Tyr Phe Lys Ala Ile Trp Asn Val Val Asn TrpLys Glu Ala 210 215 220 Ser Arg Arg Phe Asp Ala Gly Lys Ile 225 230 (2)INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GCTCTAGATCGTCGGAGCTC ATGTCGGCGA TTTTAC 36 (2) INFORMATION FOR SEQ ID NO: 33: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 33: GCGGTACCAC GCCTAGAGCA AAGTATAAAT AAGGAA 36(2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 346 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:ATATCGACTG TAGTAATATC TCAGGTCTCT GGGATAGCTG ATGGAATGTT AAGTGAATAA 60TATTGATTTA AAGTTCCTCT AGTTCCAAGC TCTTATGTAG CTTCATTTTC TATATATATA 120TATTCTATTT AGTGGTGTTG CAGGCGGTGA GCCTATCGGC CAATCATAGT AAAAAACCCG 180TTAGTTGCAA TACCCTGTTA GTTGCAAGGC GAATTCCTGG CTGATATCCT TGCAACTAAC 240GGGGTTTCTC AGTACTCGAA TTGAATATAT ATTTCGCACA AAGTTATTTC GCAAACTTGG 300GGGCCCTGGG GTCATACAAC CCAAGCCACA AGCTTTATTT AATTCG 346 (2) INFORMATIONFOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: CTATGATTGG CCGATAGG 18(2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:CCAGGCTCGC ACGCTTTC 18 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 37: CTTGCAACTA ACGGGGTT 18 (2) INFORMATION FOR SEQ ID NO: 38: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 38: TGAGAAAGAC CAAGAATG 18 (2) INFORMATION FORSEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 188 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GATTTAATGA CTACCTTGATGATACTGCCA ATATAGTTAG ATAATACAAA TCCTGGCTGC 60 CATATAACGC CCTCGCAAACGACATCTTGT TCTTATTNTC CCTCAATCGA GCTTGCCTAT 120 GCCCAAGCTT CGAACTATACGAGCATTGTA AATTGATTTT GATACGGCCT GCCATATCAG 180 ATTGACTC 188 (2)INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: TGTAGTCTGACTAGCATG 18 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 41: GGATCTTCAC CTAGATCC 18 (2) INFORMATION FOR SEQ ID NO: 42: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 42: CATAGTGTCG ACCAAGC 17 (2) INFORMATION FORSEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: CAATCGAGCT TGCCTATG 18 (2)INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: ATTTAAATGGTCCTCGGTGG ATCAAGC 27 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 45: TTAATTAATT AGTCCTGTCT GCGCTGGT 28 (2) INFORMATION FOR SEQ IDNO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 46: CGGTGGATCA AGCGGTTAAT TAATCACTCCGTACCTGAT 39 (2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 47: GCACTCGAAT GACTACT 17 (2) INFORMATION FOR SEQ ID NO: 48: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 48: CGCATCATAC TTGCGACA 18 (2) INFORMATION FORSEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: AGAAATCGGG TATCCTTTCA G 21 (2)INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1132 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 50: AACCCTGTAT GTGAGCCTGA TTCAAGACTT CGGATTTACT TTCAAGGCTTCGAATCACTG 60 TTAAGGCAGA AGAAAGTAGT ACTAATGGTT ATCAATATAT AGGAGGGCAAGCGGGAGGTT 120 ATCGCCAACG AAGAAGGAGG TTAGTCCACT ACTGCTTGGT GGCGGGTAATCTTAAGAGCA 180 CAAACTAACG GATACACAGA TCGTCAAATC CCTTCCGTCC TTTCATACATTGATGGTGAG 240 GAGTACCACG GTACTCAAGC CAAGGCCCAG TTGGTCCGCA ACTCCCAGAACACTGTCGCA 300 TACTTCAGAG ATTACCTTGG CAAGGAGTTC AAGTCGATAG ACGCCACACCATGCCATAAC 360 TCGGCGCATC CTCAGCCTCA CGAGTCTACC GTTGCTTTCT CCATTGTGGACTCTACCAAC 420 GAGACCCCCA GCACTGTCAC CGTCTCCGAG ATTGCCACCC GCCATCTCCGTCGTTTGAAG 480 CAGTCCGCCT CTGACTACCT GGGCAAGGAA GTCAATGCCG CCGTCATCACTGTCCCCACT 540 GACTTCTCCG ATGCTCAGCG CGAGGCTTTG ACCGCTTCCG CTAAGGCTGCTGGCCTTGAG 600 GTCCTCCAGC TCATCCATGA GCCTGTTGCC GCTGCCCTGG CTTACGATGCCAGGCCCGAG 660 GCTACTGTTA CTGACAAGCT TGTTGTCGTC GCCGACCTCG GTGGTACCCGATCCGACGCT 720 GCTGTTCTCG CTTGCCGTGG TGGCATGTAC AGTATCCTCG CAACTGCTCATGACTACGAG 780 TTGGGTGGAG CTTCGTTGGA CAAGATCATC ATCGACCATT TCGCCAAGGAGTTCATTAAG 840 AAGCACAAGA CCGATCCTCG CGAGAACGCT CGTGGTCTCG CCAAGTTGAAGCTTGAGGGT 900 GAGGCTGCTC GCAAGACCTT GAGCTTGGGT ACCAACGCCA GCTTGAGCATTGAGATCTTC 960 GCAGATGGCA TTGATTTCGG CTCCACTGTC AACCGTACTC GNTACGAACTTCTTTCCGGC 1020 AAGACCTTCG CCCAGTTCAC CGGCTTGATC GAGCAGGTTA TCCAGAAGGCTGGTTTGGAT 1080 GTTTTGGACA TTGACGAGGT TAGTCCCTTG TGATTTTTTT TTTTTTTTTCAG 1132 (2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 374 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 51: Asn Pro Val Cys Glu Pro Asp Ser Arg Leu ArgIle Tyr Phe Gln Gly 1 5 10 15 Phe Glu Ser Leu Leu Arg Gln Lys Lys ValVal Leu Met Val Ile Asn 20 25 30 Ile Glu Gly Lys Arg Glu Val Ile Ala AsnGlu Glu Gly Gly Ser Thr 35 40 45 Thr Ala Trp Trp Arg Val Ile Leu Arg AlaGln Thr Asn Gly Tyr Thr 50 55 60 Asp Arg Gln Ile Pro Ser Val Leu Ser TyrIle Asp Gly Glu Glu Tyr 65 70 75 80 His Gly Thr Gln Ala Lys Ala Gln LeuVal Arg Asn Ser Gln Asn Thr 85 90 95 Val Ala Tyr Phe Arg Asp Tyr Leu GlyLys Glu Phe Lys Ser Ile Asp 100 105 110 Ala Thr Pro Cys His Asn Ser AlaHis Pro Gln Pro His Glu Ser Thr 115 120 125 Val Ala Phe Ser Ile Val AspSer Thr Asn Glu Thr Pro Ser Thr Val 130 135 140 Thr Val Ser Glu Ile AlaThr Arg His Leu Arg Arg Leu Lys Gln Ser 145 150 155 160 Ala Ser Asp TyrLeu Gly Lys Glu Val Asn Ala Ala Val Ile Thr Val 165 170 175 Pro Thr AspPhe Ser Asp Ala Gln Arg Glu Ala Leu Thr Ala Ser Ala 180 185 190 Lys AlaAla Gly Leu Glu Val Leu Gln Leu Ile His Glu Pro Val Ala 195 200 205 AlaAla Leu Ala Tyr Asp Ala Arg Pro Glu Ala Thr Val Thr Asp Lys 210 215 220Leu Val Val Val Ala Asp Leu Gly Gly Thr Arg Ser Asp Ala Ala Val 225 230235 240 Leu Ala Cys Arg Gly Gly Met Tyr Ser Ile Leu Ala Thr Ala His Asp245 250 255 Tyr Glu Leu Gly Gly Ala Ser Leu Asp Lys Ile Ile Ile Asp HisPhe 260 265 270 Ala Lys Glu Phe Ile Lys Lys His Lys Thr Asp Pro Arg GluAsn Ala 275 280 285 Arg Gly Leu Ala Lys Leu Lys Leu Glu Gly Glu Ala AlaArg Lys Thr 290 295 300 Leu Ser Leu Gly Thr Asn Ala Ser Leu Ser Ile GluIle Phe Ala Asp 305 310 315 320 Gly Ile Asp Phe Gly Ser Thr Val Asn ArgThr Arg Tyr Glu Leu Leu 325 330 335 Ser Gly Lys Thr Phe Ala Gln Phe ThrGly Leu Ile Glu Gln Val Ile 340 345 350 Gln Lys Ala Gly Leu Asp Val LeuAsp Ile Asp Glu Val Ser Pro Leu 355 360 365 Phe Phe Phe Phe Phe Ser 370(2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 339 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 52: Met Ser Lys Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr SerCys Val 1 5 10 15 Ala His Phe Ala Asn Asp Arg Val Asp Ile Ile Ala AsnAsp Gln Gly 20 25 30 Asn Arg Thr Thr Pro Ser Phe Val Ala Phe Thr Asp ThrGlu Arg Leu 35 40 45 Ile Gly Asp Ala Ala Lys Asn Gln Ala Ala Met Asn ProSer Asn Thr 50 55 60 Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Asn Phe AsnAsp Pro Glu 65 70 75 80 Val Gln Ala Asp Met Lys His Phe Pro Phe Lys LeuIle Asp Val Asp 85 90 95 Gly Lys Pro Gln Ile Gln Val Glu Phe Lys Gly GluThr Lys Asn Phe 100 105 110 Thr Pro Glu Gln Ile Ser Ser Met Val Leu GlyLys Met Lys Glu Thr 115 120 125 Ala Glu Ser Tyr Leu Gly Ala Lys Val AsnAsp Ala Val Val Thr Val 130 135 140 Pro Ala Tyr Phe Asn Asp Ser Gln ArgGln Ala Thr Lys Asp Ala Gly 145 150 155 160 Thr Ile Ala Gly Leu Asn ValLeu Arg Ile Ile Asn Glu Pro Thr Ala 165 170 175 Ala Ala Ile Ala Tyr GlyLeu Asp Lys Lys Gly Lys Glu Glu His Val 180 185 190 Leu Ile Phe Asp LeuGly Gly Gly Thr Phe Asp Val Ser Leu Leu Phe 195 200 205 Ile Glu Asp GlyIle Phe Glu Val Lys Ala Thr Ala Gly Asp Thr His 210 215 220 Leu Gly GlyGlu Asp Phe Asp Asn Arg Leu Val Asn His Phe Ile Gln 225 230 235 240 GluPhe Lys Arg Lys Asn Lys Lys Asp Leu Ser Thr Asn Gln Arg Ala 245 250 255Leu Arg Arg Leu Arg Thr Ala Cys Glu Ser Gln Glu Asn Phe Val Ser 260 265270 Ser Ala Gln Thr Ser Val Glu Ile Asp Ser Lys Asn Glu Gly Ile Asp 275280 285 Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Glu Glu Leu Cys Ala Asp290 295 300 Leu Phe Arg Ser Thr Leu Asp Pro Val Glu Lys Val Leu Arg AspAla 305 310 315 320 Lys Leu Asp Lys Ser Gln Val Asp Glu Ile Val Leu ValGly Gly Ser 325 330 335 Thr Arg Ile (2) INFORMATION FOR SEQ ID NO: 53:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 560 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: Lys Thr Leu Pro PheSer Glu Asn Phe Ile Met Ala Asp Ser Glu Glu 1 5 10 15 Tyr Lys Thr ValIle Gly Ile Ser Phe Gly Asn Gln Asn Ser Ser Ile 20 25 30 Ala Phe Asn ArgAsp Gly Lys Thr Asp Val Leu Ala Asn Glu Glu Gly 35 40 45 Asn Arg Gln IlePro Ser Ile Leu Pro Tyr His Gly Asp Gln Glu Tyr 50 55 60 His Gly Val GlnAla Arg Gly Gln Leu Val Arg Asn Ala Asp Asn Ser 65 70 75 80 Val Thr AsnPhe Arg Asp Leu Leu Gly Lys Ser His Asp Glu Leu Thr 85 90 95 His His HisCys His Tyr Ser Ser Asn Pro Val Asn Val Glu Gly Gln 100 105 110 Ile GlyPhe Lys Ile Thr Val Gln Glu Gly Glu Glu Ser Asp Pro Lys 115 120 125 GluLys Ile Leu Thr Ala His Glu Ala Ser Val Arg His Leu Arg Arg 130 135 140Leu Thr Glu Ser Ala Glu Asp Phe Leu Gly Thr Lys Val Asn Gly Cys 145 150155 160 Val Met Ser Val Pro Val Tyr Phe Thr Asp Ala Gln Arg Lys Ala Leu165 170 175 Glu Ser Ala Ala Asn Glu Ala Gly Leu Pro Val Leu Gln Leu IleHis 180 185 190 Asp Pro Ala Ala Val Ile Leu Ala Leu Met Tyr Ser Glu GluVal Leu 195 200 205 Ile Asp Lys Thr Val Val Val Ala Asn Phe Gly Ala ThrArg Ser Glu 210 215 220 Val Ser Val Val Ser Val Lys Gly Gly Leu Met ThrIle Leu Ala Ser 225 230 235 240 Val His Asp Glu Asn Leu Gly Gly Glu GlnLeu Thr Asp Val Leu Val 245 250 255 Asn Phe Phe Ala Lys Glu Phe Glu LysLys Asn Gly Ile Asp Pro Arg 260 265 270 Lys Asn Ala Arg Ser Leu Val LysLeu Arg Ala Gln Cys Glu Ile Thr 275 280 285 Lys Arg Val Leu Ser Asn GlyThr Thr Ala Ser Ala Ala Val Asp Ser 290 295 300 Leu Ala Asp Gly Ile AspPhe His Ser Ser Ile Asn Arg Leu Arg Tyr 305 310 315 320 Asp Leu Ala AlaSer Ala Thr Leu Asn Arg Met Ala Asp Leu Val Thr 325 330 335 Glu Ala ValGlu Lys Ala Asn Met Glu Pro Phe Asp Ile Ser Glu Val 340 345 350 Ile LeuAla Gly Gly Ala Ser Asn Thr Pro Lys Leu Thr Ser Leu Met 355 360 365 GluSer Ile Phe Pro Glu Gln Thr Ile Ile Arg Ser Ser Ser Ser Val 370 375 380Thr Pro Leu Gln Leu Asp Pro Ser Glu Leu Thr Ala Ile Gly Ser Gly 385 390395 400 Val Gln Ala Ser Leu Ile Gly His Phe Asp Ala Ala Asp Ile Ala Ala405 410 415 Ser Thr Asp Ala Gln Val Val Asp Val Pro His Leu Thr Ala ProIle 420 425 430 Gly Ile Asn Glu Gly Glu Asn Phe Val Thr Ile Phe Asp IleGlu Thr 435 440 445 Ala Leu Pro Ala Arg Lys Thr Val Glu Val Ile Ala ProLys Glu Gly 450 455 460 Ala Ala Phe Ile Pro Ile Tyr Glu Ala Glu Arg SerVal Lys Val Thr 465 470 475 480 Lys Val Glu Pro Glu Pro Ile Asp Glu GluGlu Ala Phe Ser Asp Asp 485 490 495 Glu Glu Glu Glu Pro Glu Glu Ile LysGlu Arg Ile Ala Ile Pro Lys 500 505 510 Thr Leu Ile Ala Thr Ile Thr LeuPro Asp Val Ser Pro Asn Ala Lys 515 520 525 Ile Glu Leu Val Leu Gln IleAsp Ala Glu Gly Lys Leu Thr Ala Ser 530 535 540 Ala Arg Pro Lys Asp GlyLys Gly Thr Asn Val Arg Gly Ser Thr Ala 545 550 555 560 (2) INFORMATIONFOR SEQ ID NO: 54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: TACGGTTGAC AGTGGAGC 18(2) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:CACTGACTTC TCCGATGC 18 (2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 501 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 56: ATTTCCCAGC GTCGTCGTTG GTTGAACGGTTCTTTTGCGG CCGGTCTCTA TTCGCTCATG 60 CATTTCGGTC GGATGTACAA GAGTGGACATAACATCATCC GTATGTTCTT CTTGCACATT 120 CAGATGTTGT ACAACGTTTT CAACACTATCCTTACATGGT TCTCCCTGGC ATCTTACTGG 180 TTGACCACCA CCGTCATCAT GGACTTGGTCGGAACGCCCA GTGAGAGCAA CGGTAACAAA 240 GGATTCCCCT TCGGTAAATC GGCGACCCCTATTATCAACA CAATTGTGAA GTATGTCTAC 300 CTCGGATTGT TGCTCTTGCA GTTCATTCTCGCTCTCGGTA ACCGCCCCAA GGGATCCCGC 360 TTCTCGTACC TGACATCTTT CGTCGTATTCGGTATCATTC AAATCTACGT TGTCGTCGAC 420 GCTCTGTACT TGGTGGTTCG TGCATTCAAGGTGTTGGCGA ATTCCTCAAG TCGTTCTTCT 480 CGTCTTCCGG CGCCAGCGCC A 501 (2)INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:178 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 57: Ile Ser Gln Arg Arg Arg Trp Leu Asn Gly Ser Phe Ala Ala GlyLeu 1 5 10 15 Tyr Ser Leu Met His Phe Gly Arg Met Tyr Lys Ser Gly HisAsn Ile 20 25 30 Ile Arg Met Phe Phe Leu His Ile Gln Met Leu Tyr Asn ValPhe Asn 35 40 45 Thr Ile Leu Thr Trp Phe Ser Leu Ala Ser Tyr Trp Leu ThrThr Thr 50 55 60 Val Ile Met Asp Leu Val Gly Thr Pro Ser Glu Ser Asn GlyAsn Lys 65 70 75 80 Gly Phe Pro Phe Gly Lys Ser Ala Thr Pro Ile Ile AsnThr Ile Val 85 90 95 Lys Tyr Val Tyr Leu Gly Leu Leu Leu Leu Gln Phe IleLeu Ala Leu 100 105 110 Gly Asn Arg Pro Lys Gly Ser Arg Phe Ser Tyr LeuThr Ser Phe Val 115 120 125 Val Phe Gly Ile Ile Gln Ile Tyr Val Val ValAsp Ala Leu Tyr Leu 130 135 140 Val Val Arg Ala Phe Thr Asn Ser Asp AlaIle Asp Phe Val Thr Asp 145 150 155 160 Gln Gly Val Gly Glu Phe Leu LysSer Phe Phe Ser Ser Ser Gly Ala 165 170 175 Ser Ala (2) INFORMATION FORSEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 916 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: Met AlaTyr His Gly Ser Gly Pro Gln Ser Pro Gly Glu His Thr Tyr 1 5 10 15 AspAsp Gly His Gln Leu Arg Asp Leu Ser His Ser Asn Thr Ser Tyr 20 25 30 GluGlu Glu Ala Ser His Gly Leu Leu Ser Ser Gln Gln Ser Pro Phe 35 40 45 AlaGly Pro Phe Asp Asp Pro His Gln Gln Arg Gly Leu Thr Ala Ser 50 55 60 ProVal Gln Arg Pro Thr Ser Gly Tyr Ser Leu Thr Glu Ser Tyr Ala 65 70 75 80Pro Asp Ala Ala Tyr His Asp Pro Tyr Ser Ala Asn Gln Ser Val Tyr 85 90 95Ser Gly His Ser Glu Asn Pro Ala Ala Ala Phe Gly Val Pro Gly Arg 100 105110 Val Ala Ser Pro Tyr Ala Arg Ser Glu Thr Ser Ser Thr Glu Ala Trp 115120 125 Arg Gln Arg Gln Ala Gly Ala Arg Arg Gly Gly Asn Gly Leu Arg Arg130 135 140 Tyr Ala Thr Arg Lys Val Lys Leu Val Gln Gly Ser Val Leu SerVal 145 150 155 160 Asp Tyr Pro Val Pro Ser Ala Ile Gln Asn Ala Ile GlnAla Lys Tyr 165 170 175 Arg Asn Asp Leu Glu Gly Gly Ser Glu Glu Phe ThrHis Met Arg Tyr 180 185 190 Thr Ala Ala Thr Cys Asp Pro Asn Glu Phe ThrLeu His Asn Gly Tyr 195 200 205 Asn Leu Arg Pro Ala Met Tyr Asn Arg HisThr Glu Leu Leu Ile Ala 210 215 220 Ile Thr Tyr Tyr Asn Glu Asp Lys ThrLeu Thr Ala Arg Thr Leu His 225 230 235 240 Gly Val Met Gln Asn Ile ArgAsp Ile Val Asn Leu Lys Lys Ser Glu 245 250 255 Phe Trp Asn Lys Gly GlyPro Ala Trp Gln Lys Ile Val Val Cys Leu 260 265 270 Val Phe Asp Gly IleAsp Pro Cys Asp Lys Asp Thr Leu Asp Val Leu 275 280 285 Ala Thr Val GlyIle Tyr Gln Asp Gly Val Met Lys Arg Asp Val Asp 290 295 300 Gly Lys GluThr Val Ala His Ile Phe Glu Tyr Thr Thr Gln Leu Ser 305 310 315 320 ValThr Pro Asn Gln Gln Leu Ile Arg Pro Thr Asp Asp Gly Pro Ser 325 330 335Thr Leu Pro Pro Val Gln Met Met Phe Cys Leu Lys Gln Lys Asn Ser 340 345350 Lys Lys Ile Asn Ser His Arg Trp Leu Phe Asn Ala Phe Gly Arg Ile 355360 365 Leu Asn Pro Glu Val Cys Ile Leu Leu Asp Ala Gly Thr Lys Pro Gly370 375 380 Pro Lys Ser Leu Leu Tyr Leu Trp Glu Ala Phe Tyr Asn Asp LysAsp 385 390 395 400 Leu Gly Gly Ala Cys Gly Glu Ile His Ala Met Leu GlyLys Gly Trp 405 410 415 Lys Lys Leu Leu Asn Pro Leu Val Ala Ala Gln AsnPhe Glu Tyr Lys 420 425 430 Ile Ser Asn Ile Leu Asp Lys Pro Leu Glu SerSer Phe Gly Tyr Val 435 440 445 Ser Val Leu Pro Gly Ala Phe Ser Ala TyrArg Phe Arg Ala Ile Met 450 455 460 Gly Arg Pro Leu Glu Gln Tyr Phe HisGly Asp His Thr Leu Ser Lys 465 470 475 480 Gln Leu Gly Lys Lys Gly IleGlu Gly Met Asn Ile Phe Lys Lys Asn 485 490 495 Met Phe Leu Ala Glu AspArg Ile Leu Cys Phe Glu Leu Val Ala Lys 500 505 510 Ala Gly Ser Lys TrpHis Leu Ser Tyr Val Lys Ala Ser Lys Gly Glu 515 520 525 Thr Asp Val ProGlu Gly Ala Pro Glu Phe Ile Ser Gln Arg Arg Arg 530 535 540 Trp Leu AsnGly Ser Phe Ala Ala Gly Ile Tyr Ser Leu Met His Phe 545 550 555 560 GlyArg Met Tyr Lys Ser Gly His Asn Ile Val Arg Met Phe Phe Leu 565 570 575His Leu Gln Met Leu Tyr Asn Trp Phe Ser Thr Phe Leu Thr Trp Phe 580 585590 Ser Leu Ala Ser Tyr Trp Leu Thr Thr Ser Val Ile Met Asp Leu Val 595600 605 Gly Thr Pro Ser Ser Ser Asn Gly Tyr Thr Ala Phe Pro Phe Gly Lys610 615 620 Thr Ala Thr Pro Ile Ile Asn Thr Leu Val Lys Tyr Ile Tyr LeuAla 625 630 635 640 Phe Leu Leu Leu Gln Phe Ile Leu Ala Leu Gly Asn ArgPro Lys Gly 645 650 655 Ser Lys Leu Ser Tyr Leu Ala Ser Phe Val Ala PheGly Ile Ile Gln 660 665 670 Leu Tyr Val Val Val Asp Ala Leu Tyr Leu ValVal Arg Ala Phe Thr 675 680 685 Gly Gly Ala Pro Met Asp Phe Asn Thr AspAsp Gly Ile Gly Ala Phe 690 695 700 Leu Ser Ser Phe Phe Gly Ser Ser GlyAla Gly Ile Ile Ile Ile Ala 705 710 715 720 Leu Ala Ala Thr Phe Gly LeuTyr Phe Val Ala Ser Phe Met Tyr Leu 725 730 735 Asp Pro Trp His Met PheThr Ser Phe Pro Ala Tyr Met Ala Val Gln 740 745 750 Ser Ser Tyr Ile AsnIle Leu Asn Val Tyr Ala Phe Ser Asn Trp His 755 760 765 Asp Val Ser TrpGly Thr Lys Gly Ser Asp Lys Ala Asp Ala Leu Pro 770 775 780 Ser Ala LysThr Thr Gly Gly Lys Gly Glu Glu Ala Val Ile Glu Glu 785 790 795 800 IleAsp Lys Pro Gln Ala Asp Ile Asp Ser Gln Phe Glu Ala Thr Val 805 810 815Lys Arg Ala Leu Thr Pro Tyr Val Pro Pro Glu Glu Lys Glu Glu Lys 820 825830 Ser Leu Asp Asp Ser Tyr Lys Ser Phe Arg Thr Arg Leu Val Thr Leu 835840 845 Trp Leu Phe Ser Asn Gly Leu Leu Ala Val Cys Ile Thr Ser Glu Gly850 855 860 Leu Asp Lys Phe Gly Phe Thr Asn Thr Ser Thr Glu Arg Thr SerArg 865 870 875 880 Phe Phe Gln Ala Leu Leu Trp Ser Asn Ala Val Val AlaLeu Ile Arg 885 890 895 Phe Ile Gly Ala Thr Trp Phe Leu Gly Lys Thr GlyLeu Leu Cys Cys 900 905 910 Phe Ala Arg Arg 915 (2) INFORMATION FOR SEQID NO: 59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 911 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: Met AlaTyr Gln Gly Ser Gly Ser His Ser Pro Pro His Tyr Asp Asp 1 5 10 15 AsnGly His Arg Leu Gln Asp Leu Pro His Gly Ser Tyr Glu Glu Glu 20 25 30 AlaSer Arg Gly Leu Leu Ser His Gln Gln Gly Pro Phe Thr Gly Pro 35 40 45 PheAsp Asp Pro Gln Gln His Gly Ser Ser Thr Thr Arg Pro Val Ser 50 55 60 GlyTyr Ser Leu Ser Glu Thr Tyr Ala Pro Glu Ala Ala Tyr His Asp 65 70 75 80Pro Tyr Thr Gln Pro Ser Pro Gly Ser Val Tyr Ser Ala Gln Ser Ala 85 90 95Glu Asn Pro Ala Ala Ala Phe Gly Val Pro Gly Arg Val Ala Ser Pro 100 105110 Tyr Ala Arg Ser Asp Thr Ser Ser Thr Glu Ala Trp Arg Gln Arg Gln 115120 125 Ala Pro Gly Gly Gly Pro Gly Gly Leu Arg Arg Tyr Ala Thr Arg Lys130 135 140 Val Lys Leu Val Gln Gly Ser Val Leu Ser Val Asp Tyr Pro ValPro 145 150 155 160 Ser Ala Ile Gln Asn Ala Ile Gln Ala Lys Tyr Arg AsnAsp Leu Glu 165 170 175 Gly Gly Ser Glu Glu Phe Thr His Met Arg Tyr ThrAla Ala Thr Cys 180 185 190 Asp Pro Asn Glu Phe Thr Leu His Asn Gly TyrAsn Leu Arg Pro Ala 195 200 205 Met Tyr Asn Arg His Thr Glu Leu Leu IleAla Ile Thr Tyr Tyr Asn 210 215 220 Glu Asp Lys Thr Leu Thr Ser Arg ThrLeu His Gly Val Met Gln Asn 225 230 235 240 Ile Arg Asp Ile Val Asn LeuLys Lys Ser Glu Phe Trp Asn Lys Gly 245 250 255 Gly Pro Ala Trp Gln LysIle Val Val Cys Leu Val Phe Asp Gly Ile 260 265 270 Asp Pro Cys Asp LysAsp Thr Leu Asp Val Leu Ala Thr Ile Gly Val 275 280 285 Tyr Gln Asp GlyVal Met Lys Arg Asp Val Asp Gly Lys Glu Thr Val 290 295 300 Ala His IlePhe Glu Tyr Thr Thr Gln Leu Ser Val Thr Pro Asn Gln 305 310 315 320 GlnLeu Ile Arg Pro Thr Asp Asp Gly Pro Ser Thr Leu Leu Pro Ser 325 330 335Lys Met Met Phe Cys Leu Lys Gln Lys Asn Ser Lys Lys Ile Asn Ser 340 345350 His Arg Trp Leu Phe Asn Ala Phe Gly Arg Ile Leu Asn Pro Glu Val 355360 365 Cys Ile Leu Leu Asp Ala Gly Thr Lys Pro Gly Pro Lys Ser Leu Leu370 375 380 Ser Leu Trp Glu Ala Phe Tyr Asn Asp Lys Asp Leu Gly Gly AlaCys 385 390 395 400 Gly Glu Ile His Ala Met Leu Gly Lys Gly Trp Lys AsnLeu Ile Asn 405 410 415 Pro Leu Val Ala Ala Gln Asn Phe Glu Tyr Lys IleSer Asn Ile Leu 420 425 430 Asp Lys Pro Leu Glu Ser Ser Phe Gly Tyr ValSer Val Leu Pro Gly 435 440 445 Ala Phe Ser Ala Tyr Arg Phe Arg Ala IleMet Gly Arg Pro Leu Glu 450 455 460 Gln Tyr Phe His Gly Asp His Thr LeuSer Lys Gln Leu Gly Lys Lys 465 470 475 480 Gly Ile Glu Gly Met Asn IlePhe Lys Lys Asn Met Phe Leu Ala Glu 485 490 495 Asp Arg Ile Leu Cys PheGlu Leu Val Ala Lys Ala Gly Ser Lys Trp 500 505 510 His Leu Thr Tyr ValLys Ala Ser Lys Ala Glu Thr Asp Val Pro Glu 515 520 525 Gly Ala Pro GluPhe Ile Ser Gln Arg Arg Arg Trp Leu Asn Gly Ser 530 535 540 Phe Ala AlaGly Ile Tyr Ser Leu Met His Phe Gly Arg Met Tyr Lys 545 550 555 560 SerGly His Asn Ile Val Arg Met Phe Phe Leu His Ile Gln Met Leu 565 570 575Tyr Asn Ile Phe Ser Thr Val Leu Thr Trp Phe Ser Leu Ala Ser Tyr 580 585590 Trp Leu Thr Thr Thr Val Ile Met Asp Leu Val Gly Thr Pro Ser Asp 595600 605 Asn Asn Gly Asn Lys Ala Phe Pro Phe Gly Lys Thr Ala Thr Pro Ile610 615 620 Ile Asn Thr Ile Val Lys Tyr Val Tyr Leu Gly Phe Leu Leu LeuGln 625 630 635 640 Phe Ile Leu Ala Leu Gly Asn Arg Pro Lys Gly Ser LysPhe Ser Tyr 645 650 655 Leu Ala Ser Phe Val Val Phe Gly Ile Ile Gln ValTyr Val Val Ile 660 665 670 Asp Ala Leu Tyr Leu Val Val Arg Ala Phe SerGly Ser Ala Pro Met 675 680 685 Asp Phe Thr Thr Asp Gln Gly Val Gly GluPhe Leu Lys Ser Phe Phe 690 695 700 Ser Ser Ser Gly Ala Gly Ile Ile IleIle Ala Leu Ala Ala Thr Phe 705 710 715 720 Gly Leu Tyr Phe Val Ala SerPhe Met Tyr Leu Asp Pro Trp His Met 725 730 735 Phe Thr Ser Phe Pro AlaTyr Met Cys Val Gln Ser Ser Tyr Ile Asn 740 745 750 Ile Leu Asn Val TyrAla Phe Ser Asn Trp His Asp Val Ser Trp Gly 755 760 765 Thr Lys Gly SerAsp Lys Ala Asp Ala Leu Pro Ser Ala Lys Thr Thr 770 775 780 Lys Asp GluGly Lys Glu Val Val Ile Glu Glu Ile Asp Lys Pro Gln 785 790 795 800 AlaAsp Ile Asp Ser Gln Phe Glu Ala Thr Val Lys Arg Ala Leu Thr 805 810 815Pro Tyr Val Pro Pro Val Glu Lys Glu Glu Lys Thr Leu Glu Asp Ser 820 825830 Tyr Lys Ser Phe Arg Thr Arg Leu Val Thr Phe Trp Ile Phe Ser Asn 835840 845 Ala Phe Leu Ala Val Cys Ile Thr Ser Asp Gly Val Asp Lys Phe Gly850 855 860 Phe Thr Asn Ser Ala Thr Asp Arg Thr Gln Arg Phe Phe Gln AlaLeu 865 870 875 880 Leu Trp Ser Asn Ala Val Val Ala Leu Phe Arg Phe IleGly Ala Cys 885 890 895 Trp Phe Leu Gly Lys Thr Gly Leu Met Cys Cys PheAla Arg Arg 900 905 910 (2) INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 60: CACCAAGTCA GAGCGTC 17 (2) INFORMATION FOR SEQ ID NO: 61: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 61: GGCCTTYGAY GAYCCCA 17 (2) INFORMATION FORSEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: GGGCCGTTTG ACAATCCGCA T 21 (2)INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:251 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 63: AGGGCCACAG CNTGTGCTAA GCGCCTTGAC GGGGACCCTG GGACTTTCAAGTCTCCTTGG 60 ACCCGGAATT GAATCCTCAC AGAACAGCTT TCAACACTGC TCTAAGGCTGAACTGAGCTG 120 CGCGACTCCG TATCATGGCC AAGACAAATG CTGCTTCAAC TATCCCGGGGGGCAGTTCCT 180 TCAATCGCTG TTTTGGGACG CCGACCCGGC CATTGGACCG GAAGATTCCTGGACTATCCA 240 TGGCTTATGG T 251 (2) INFORMATION FOR SEQ ID NO: 64: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 83 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: Gly Pro Gln Xaa Val LeuSer Ala Leu Thr Gly Thr Leu Gly Leu Ser 1 5 10 15 Ser Leu Leu Gly ProGly Ile Glu Ser Ser Gln Asn Ser Phe Gln His 20 25 30 Cys Ser Lys Ala GluLeu Ser Cys Ala Thr Pro Tyr His Gly Gln Asp 35 40 45 Lys Cys Cys Phe AsnTyr Pro Gly Gly Gln Phe Leu Gln Ser Leu Phe 50 55 60 Trp Asp Ala Asp ProAla Ile Gly Pro Glu Asp Ser Trp Thr Ile His 65 70 75 80 Gly Leu Trp (2)INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:83 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 65: Gly Pro Gln Xaa Val Leu Ser Ala Leu Thr Gly Thr Leu Gly LeuSer 1 5 10 15 Ser Leu Leu Gly Pro Gly Ile Glu Ser Ser Gln Asn Ser PheGln His 20 25 30 Cys Ser Lys Ala Glu Leu Ser Cys Ala Thr Pro Tyr His GlyGln Asp 35 40 45 Lys Cys Cys Phe Asn Tyr Pro Gly Gly Gln Phe Leu Gln SerLeu Phe 50 55 60 Trp Asp Ala Asp Pro Ala Ile Gly Pro Glu Asp Ser Trp ThrIle His 65 70 75 80 Gly Leu Trp (2) INFORMATION FOR SEQ ID NO: 66: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 214 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 66: CTACTGAACG CTTAAAGGTG CTTAAGGAGC AACTTCATATTATGCGCGAC CAACGGATCC 60 AGGAAGTCTT GAGCAATAAG AAGGGTCGAA CGCAGCACGGACACTCGCAC AAGCCGACCG 120 GTTTTGGGGG ACTCAACGGT TCTCGGCTAA AGGAGGCCTTTGTGGGACGT CGAATCGGGA 180 AGAATTCCAA GGCATTGGCC GAATTGGCCA CCCC 214 (2)INFORMATION FOR SEQ ID NO: 67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: GTTCTATTGAGATACGCG 18 (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 68: ACAAGCCGAC CGGTTTTG 18 (2) INFORMATION FOR SEQ ID NO: 69: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 69: CGATAAGGAC TCCAAGAG 18 (2) INFORMATION FORSEQ ID NO: 70: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: GTCGCGCATA ATATGAAG 18 (2)INFORMATION FOR SEQ ID NO: 71: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:336 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: AGCACCTATAATCTATGCTG TCCCACTATC ACACATCTAT ATGTTGTACA AGCCTGATAC 60 AATCAATAATGATGTAATAA TTGACTCTGG AAAGTTGGCT ATAAAACTCA CCATACAAGT 120 CCAGATAACCCTGCCAAACT CCACTCCCAG GGCATTAATC TTCATTTATA TCGACCAGCC 180 ATACCTATGGTCAAATCACA CGCAACGCCA CAGATATATA TTTGAATCAA ATTTCTCTTT 240 TGAAGAAGAAAGGGTGGTTT ATGAGGAAGA ATATCCCAAT ATGCCAATCT GACTGTTCCG 300 GATTGGAATAATGCACAAGC TTCGAATATA AATATA 336 (2) INFORMATION FOR SEQ ID NO: 72: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 72: CTTCCTCATA AACCACCC 18 (2) INFORMATION FORSEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: AACTGACAGG ACAAGACC 18 (2)INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: GACTTGCATCACTTCCTC 18 (2) INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 75: TGAAGCTGAG AGTAGGTG 18 (2) INFORMATION FOR SEQ ID NO: 76: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 281 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 76: CAAGGGAACG GGAATAAAAT ACACATAACA AAGGATTCGAAGAAAGAAAA AAAAAGGGGG 60 GGAGGTGTGT CCAAGAGGAA AGAAGAAAAA AAATTTAATTTCGCCACCCT ATCGCGGAGT 120 GTTCCGCCTT CAGGAGAGAT AGAAAAGAGG AGGGAGAAGGGAGAAGGAAA AAAAAAAACA 180 GAATTCCCAC AGACAAGGAA AGCTTAACCG GGTCACGAAAAAGCACAATA CAGGTGAACA 240 ACTGAGGGGA AGGGGGCCAA AAAGAAAAAA ATAATTCCTA A281 (2) INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:GTTTCGGTAT TGTCACTG 18 (2) INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO: 78: ACAGGTGAAC AACTGAGG 18 (2) INFORMATION FOR SEQ ID NO: 79: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 79: CGACCAAACT AGACAAGC 18 (2) INFORMATION FORSEQ ID NO: 80: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:CTTTCCTCTT GGACACAC 18

What is claimed is:
 1. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for productionof the polypeptide, wherein (i) the mutant cell is related to a parentcell, which comprises a first DNA sequence encoding the polypeptide, bythe introduction of a nucleic acid construct into the genome of theparent cell at a locus which is not within the first DNA sequence, notwithin a second DNA sequence encoding a protein that negativelyregulates transcription, translation or secretion of the polypeptide,and not within a third DNA sequence encoding a protease capable ofhydrolyzing the polypeptide under the conditions; and (ii) the mutantcell produces more of the polypeptide than the parent cell when bothcells are cultivated under the conditions; and (b) recovering thepolypeptide.
 2. The method of claim 1, wherein the nucleic acidconstruct has less than 40% homology with the first DNA sequence.
 3. Themethod of claim 1, wherein the nucleic acid construct has less than 40%homology with the locus.
 4. The method of claim 1, wherein the locus ison a different chromosome than the first DNA sequence or on the samechromosome but at least 3,000 bps from the 5′ or 3′ terminus of thefirst DNA sequence.
 5. A method of producing a polypeptide, comprising(A) cultivating a mutant cell under conditions conducive for productionof the polypeptide, wherein (i) the mutant cell is related to a parentcell, which comprises a first DNA sequence encoding the polypeptide, bythe introduction of a nucleic acid construct into the genome of theparent cell at a locus which is not within the first DNA sequence,wherein the introduction of the nucleic acid construct disrupts a geneencoding an oxidoreductase, a transferase, a hydrolase, a lyase, anisomerase, a ligase, or regulatory or control sequences thereof, otherthan a gene encoding a protease which is capable of hydrolyzing thepolypeptide under the conditions; and (ii) the mutant cell produces moreof the polypeptide than the parent cell when both cells are cultivatedunder the conditions; and (B) recovering the polypeptide.
 6. A method ofproducing a polypeptide, comprising (a) cultivating a mutant cell underconditions conducive for production of the polypeptide, wherein (i) themutant cell is related to a parent cell, which comprises a first DNAsequence encoding the polypeptide, by the introduction of a nucleic acidconstruct into the genome of the parent cell at a locus which is notwithin the first DNA sequence, not within a second DNA sequence encodinga protein that negatively regulates transcription of the polypeptide,and not within a third DNA sequence encoding a protease capable ofhydrolyzing the polypeptide under the conditions; and (ii) the mutantcell expresses more of the polypeptide than the parent cell when bothcells are cultivated under the conditions; and (b) recovering thepolypeptide.
 7. The method of claim 6, wherein the nucleic acidconstruct has less than 40% homology with the first DNA sequence.
 8. Themethod of claim 6, wherein the nucleic acid construct has less than 40%homology with the locus.
 9. The method of claim 6, wherein the locus ison a different chromosome than the first DNA sequence or on the samechromosome but at least 3,000 bps from the 5′ or 3′ terminus of thefirst DNA sequence.
 10. A method of producing a polypeptide, comprising(A) cultivating a mutant cell under conditions conducive for productionof the polypeptide, wherein (i) the mutant cell is related to a parentcell, which comprises a first DNA sequence encoding the polypeptide, bythe introduction of a nucleic acid construct into the genome of theparent cell at a locus which is not within the first DNA sequence,wherein the introduction of the nucleic acid construct disrupts a geneencoding an oxidoreductase, a transferase, a hydrolase, a lyase, anisomerase, a ligase, or regulatory or control sequences thereof, otherthan a gene encoding a protease which is capable of hydrolyzing thepolypeptide under the conditions; and (ii) the mutant cell expressesmore of the polypeptide than the parent cell when both cells arecultivated under the conditions; and (B) recovering the polypeptide. 11.A method of producing a polypeptide, comprising (a) cultivating a mutantcell under conditions conducive for production of the polypeptide,wherein (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, not within a second DNAsequence encoding a protein that negatively regulates translation of thepolypeptide, and not within a third DNA sequence encoding a proteasecapable of hydrolyzing the polypeptide under the conditions; and (ii)the mutant cell synthesizes more of the polypeptide than the parent cellwhen both cells are cultivated under the conditions; and (b) recoveringthe polypeptide.
 12. The method of claim 11, wherein the nucleic acidconstruct has less than 40% homology with the first DNA sequence. 13.The method of claim 11, wherein the nucleic acid construct has less than40% homology with the locus.
 14. The method of claim 11, wherein thelocus is on a different chromosome than the first DNA sequence or on thesame chromosome but at least 3,000 bps from the 5′ or 3′ terminus of thefirst DNA sequence.
 15. A method of producing a polypeptide, comprising(A) cultivating a mutant cell under conditions conducive for productionof the polypeptide, wherein (i) the mutant cell is related to a parentcell, which comprises a first DNA sequence encoding the polypeptide, bythe introduction of a nucleic acid construct into the genome of theparent cell at a locus which is not within the first DNA sequence,wherein the introduction of the nucleic acid construct disrupts a geneencoding an oxidoreductase, a transferase, a hydrolase, a lyase, anisomerase, a ligase, or regulatory or control sequences thereof, otherthan a gene encoding a protease which is capable of hydrolyzing thepolypeptide under the conditions; and (ii) the mutant cell synthesizesmore of the polypeptide than the parent cell when both cells arecultivated under the conditions; and (B) recovering the polypeptide. 16.A method of producing a polypeptide, comprising (a) cultivating a mutantcell under conditions conducive for production of the polypeptide,wherein (i) the mutant cell is related to a parent cell, which comprisesa first DNA sequence encoding the polypeptide, by the introduction of anucleic acid construct into the genome of the parent cell at a locuswhich is not within the first DNA sequence, not within a second DNAsequence encoding a protein that negatively regulates secretion of thepolypeptide, and not within a third DNA sequence encoding a proteasecapable of hydrolyzing the polypeptide under the conditions; and (ii)the mutant cell secretes more of the polypeptide than the parent cellwhen both cells are cultivated under the conditions; (b) recovering thepolypeptide.
 17. The method of claim 16, wherein the nucleic acidconstruct has less than 40% homology with the first DNA sequence. 18.The method of claim 16, wherein the nucleic acid construct has less than40% homology with the locus.
 19. The method of claim 16, wherein thelocus is on a different chromosome than the first DNA sequence or on thesame chromosome but at least 3,000 bps from the 5′ or 3′ terminus of thefirst DNA sequence.
 20. A method of producing a polypeptide, comprising(A) cultivating a mutant cell under conditions conducive for productionof the polypeptide, wherein (i) the mutant cell is related to a parentcell, which comprises a first DNA sequence encoding the polypeptide, bythe introduction of a nucleic acid construct into the genome of theparent cell at a locus which is not within the first DNA sequence,wherein the introduction of the nucleic acid construct disrupts a geneencoding an oxidoreductase, a transferase, a hydrolase, a lyase, anisomerase, a ligase, or regulatory or control sequences thereof, otherthan a gene encoding a protease which is capable of hydrolyzing thepolypeptide under the conditions; and (ii) the mutant cell secretes moreof the polypeptide than the parent cell when both cells are cultivatedunder the conditions; and (B) recovering the polypeptide.
 21. A methodof producing a polypeptide, comprising (a) cultivating a mutant cellunder conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a firstDNA sequence encoding the polypeptide, by the random integration of anucleic acid construct into the genome of the parent cell at a locuswherein the nucleic acid construct is not homologous with the locus andwherein the locus is not within the first DNA sequence nor within asecond DNA sequence encoding a protease capable of hydrolyzing thepolypeptide under the conditions; and (ii) the mutant cell produces moreof the polypeptide than the parent cell when both cells are cultivatedunder the conditions; and (b) recovering the polypeptide.
 22. The methodof claim 1, wherein the nucleic acid construct is introduced byrestriction enzyme-mediated integration.
 23. The method of claim 1,wherein the nucleic acid construct comprises a selectable marker. 24.The method of claim 23, wherein the selectable marker is amdS, argB,bar, hygB, niaD, pyrG, sC, or trpC.
 25. The method of claim 1, whereinthe parent cell is a mammalian cell.
 26. The method of claim 1, whereinthe parent cell is a bacterial cell.
 27. The method of claim 1, whereinthe parent cell is a fungal cell.
 28. The method of claim 27, whereinthe fungal cell is a filamentous fungal cell.
 29. The method of claim28, wherein the filamentous fungal cell is selected from the groupconsisting of Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia,Tolypocladium, and Trichodenna.
 30. The method of claim 27, wherein thefungal cell is a yeast cell.
 31. The method of claim 1, wherein thepolypeptide is a recombinant polypeptide.
 32. The method of claim 1,wherein the polypeptide is a heterologous polypeptide.
 33. The method ofclaim 1, wherein the polypeptide is a hormone, a hormone variant, anenzyme, a receptor or portions thereof, an antibody or portions thereof,or a reporter.
 34. The method of claim 33, wherein the polypeptide is anoxidoreductase. a transferase, a hydrolase, a lyase, an isomerase, or aligase.
 35. The method of claim 34, wherein the polypeptide is anaminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, acatalase, a cellulase, a chitinase, a cutinase, a deoxyribonuclease, anesterase, an alpha-galactosidase, a beta-galactosidase, a glucoamylase,an alpha-glucosidase, a beta-glucosidase, an invertase, a laccase, alipase, a mannosidase, a mutanase, an oxidase, a pectinolytic enzyme, aperoxidase, a phytase, a polyphenoloxidase, a proteolytic enzyme, aribonuclease, or a xylanase.
 36. The method of claim 1, wherein themutant cell has an increased uptake of an inorganic cofactor compared tothe parent cell.
 37. The method of claim 1, wherein the mutant cell hasa more desirable morphology than the parent cell.
 38. The method ofclaim 1, wherein the mutant cell produces higher yields of one or moresecreted proteins than the parent cell.
 39. The method of claim 1,wherein the mutant cell which has lost its ability to synthesize one ormore essential metabolites.
 40. The method of claim 1, wherein aphenotype of the mutant cell is observed only under certain conditions.41. The method of claim 1, wherein the mutant cell exhibits an alteredgrowth rate relative to the parent cell.
 42. The method of claim 1,wherein the growth of the mutant cell is not inhibited by theoverproduction of a desired polypeptide or metabolite when grown underconditions that induce high level production of the polypeptide ormetabolite.
 43. The method of claim 1, wherein the mutant cell is ableto tolerate lower oxygen conditions than the parent cell.
 44. The methodof claim 1, wherein the mutant cell exhibits altered production of atranscriptional activator of a promoter than the parent cell.
 45. Themethod of claim 1, wherein the mutant cell has a mutation in on e ormore of the genes of the signal transduction pathway of the parent cell.46. The method of claim 1, wherein the mutant cell does not recognizeand erroneously splice a cryptic intron.
 47. The method of claim 1,wherein the nucleic acid construct is pDSY109, pDSY112, pMT1936,pDSY138, pDSY162, pDSY163, pDSY141, pSMO1204, pSMOH603, p4-8.1, p7-14.1,pHB220, pSMO717, pSMO321, pHowB571 or pSMO810.
 48. The method of claim1, wherein the locus is SEQ ID NO:9, SEQ ID NO: 16, SEQ ID NO:25, SEQ IDNO:29, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:56, SEQ IDNO:63, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:76, or a fragment thereof.49. The method of claim 1, wherein the locus encodes a glucosetransporter, mannitol-1-phosphate dehydrogenase, chitin synthase, heatshock protein, manganese superoxide dismutase, or a gene required foractivation of pacC,
 50. The method of claim 49, wherein the locus is apalB gene.
 51. A method of producing a polypeptide, comprising (a)cultivating a mutant cell under conditions conducive for production ofthe polypeptide, wherein (i) the mutant cell is related to a parentcell, which comprises a first DNA sequence encoding the polypeptide, bythe introduction of a nucleic acid construct into the genome of theparent cell at a locus which is not within the first DNA sequence and asecond DNA sequence encoding a protein that positively regulatestranscription, translation or secretion of the polypeptide; and (ii) themutant cell produces less of the polypeptide than the parent cell whenboth cells are cultivated under the conditions; and (b) recovering thepolypeptide.
 52. A method of producing a metabolite, comprising (A)cultivating a mutant cell under conditions conducive for production ofthe metabolite, wherein (i) the mutant cell is related to a parent cell,which comprises one or more first DNA sequences encoding firstpolypeptides in the biosynthetic pathway of the metabolite, by theintroduction of a nucleic acid construct into the genome of the parentcell at a locus which is not within (a) the first DNA sequences, (b) asecond DNA sequence encoding a protein that negatively regulatestranscription, translation or secretion of the first polypeptides, (c) athird DNA sequence encoding a protease capable of hydrolyzing any of thefirst polypeptides under the conditions, and (d) one or more fourth DNAsequences encoding a second polypeptide in the second biosyntheticpathway of a second metabolite wherein the biosynthetic pathway and thesecond biosynthetic pathway involve the production of the sameintermediate and the second polypeptide catalyzes a step after theproduction of the intermediate; and (ii) the mutant cell produces moreof the metabolite than the parent cell when both cells are cultivatedunder the conditions; and (B) recovering the metabolite.
 53. A method ofproducing a first polypeptide, comprising (a) forming a mutant cell byintroducing a nucleic acid construct into the genome of the parent cell,which comprises a first DNA sequence encoding the polypeptide, at alocus which is not within the first DNA sequence, a second DNA sequenceencoding a protein that negatively regulates transcription, translationor secretion of a second polypeptide, and a third DNA sequence encodinga protease capable of hydrolyzing the polypeptide under conditionsconducive to the production of the first polypeptide; (b) isolating themutant cell which produces more of the polypeptide than the parent cellwhen both cells are cultivated under the conditions; (c) identifying thelocus wherein the nucleic acid construct has been integrated; (d)producing a cell in which a corresponding locus has been disrupted; (e)culturing the cell under the conditions; and (f) recovering the firstpolypeptide.
 54. A method of producing a polypeptide, comprising (a)cultivating a mutant cell under conditions conducive for production ofthe polypeptide, wherein (i) the mutant cell is related to a parentcell, which comprises a first DNA sequence encoding the polypeptide, bythe introduction of a nucleic acid construct into the genome of theparent cell at a locus which is not within the first DNA sequence and asecond DNA sequence encoding a protease capable of hydrolyzing thepolypeptide under the conditions, wherein the introduction of thenucleic acid construct specifically enhances transcription, translationor secretion of the polypeptide; and (ii) the mutant cell produces moreof the polypeptide than the parent cell when both cells are cultivatedunder the conditions; and (b) recovering the polypeptide.